Some Interesting Uses of DNA Forensic Identification
Identifying September 11th Victims Identifying the victims of the September 11, 2001, World Trade Center attack presented a unique forensic challenge because the number and identity of the victims were unknown and many victims were represented only by bone and tissue fragments. At the time of the attack, no systems were in place for rapidly identifying victims in disasters with more than 500 fatalities. The National Institutes of Justice assembled a panel of experts from the National Institutes of Health and other institutions to develop processes to identify victims using DNA collected at the site. Panel members produced forms and kits needed to enable the medical examiner’s office to collect reference DNA from victims’ previously stored medical specimens. These specimens were collected and entered into a database. The medical examiner's office also received about 20,000 pieces of human remains from the World Trade Center site, and a database of the victims’ DNA profiles was created. New information technology infrastructure was developed for data transfer between the state police and medical examiner’s office and to interconnect the databases and analytical tools used by panel members. In 2005 the search was declared at an end because many of the unidentified remains were too small or too damaged to be identified by the DNA extraction methods available at that time. Remains of only 1585, of the 2792 people known to have died had been identified. In 2007, the medical examiner's office reopened the search after the Bode Technology Group developed a new methodology of DNA extraction that required much less sample material than previously necessary. The victim DNA database and the new methods have allowed more victims to be identified, and further identifications will be possible as forensic DNA technology improves.
The DNA Shoah ProjectThe DNA Shoah Project is a genetic database of people who lost family during the Holocaust. The database will serve to reunite families separated during wartime and aid in identifying victims who remain buried anonymously throughout Europe.
Disappeared Children in ArgentinaNumerous people (known as "the Disappeared") were kidnapped and murdered in Argentina in the 1970s. Many were pregnant. Their children were taken at birth and, along with other kidnapped children, were raised by their kidnappers. The grandparents of these children have been looking for them for many years. Read an article about a DNA researcher who has been helping them.
Tomb of the Unknowns
Son of Louis XVI and Marie AntionettePARIS, Apr 19, 2000 (Reuters) -- Scientists cracked one of the great mysteries of European history by using DNA tests to prove that the son of executed French King Louis XVI and Marie-Antoinette died in prison as a child. Royalists have argued for 205 years over whether Louis-Charles de France perished in 1795 in a grim Paris prison or managed to escape the clutches of the French Revolution. In December 1999, the presumed heart of the child king was removed from its resting place to enable scientists to compare its DNA makeup with samples from living and dead members of the royal family -- including a lock of his mother Marie-Antoinette's hair.
The Murdered Nicholas Romanov, the Last Czar of Russia, and His Family
Peruvian Ice MaidenThe Ice Maiden was a 12-to-14-year old girl sacrificed by Inca priests 500 years ago to satisfy the mountain gods of the Inca people. She was discovered in 1995 by climbers on Mt. Ampato in the Peruvian Andes. She is perhaps the best preserved mummy found in the Andes because she was in a frozen state. Analysis of the Ice Maiden's DNA offers a wonderful opportunity for understanding her genetic origin. If we could extract mitochondrial DNA from the Ice Maiden's tissue and successfully amplify and sequence it, then we could begin to trace her maternal line of descent and possibly locate past and current relatives.
African Lemba Tribesmen In southern Africa, a people known as the Lemba heed the call of the shofar. They have believed for generations that they are Jews, direct descendants of the biblical patriarchs Abraham, Isaac, and Jacob. However unlikely the Lemba's claims may seem, modern science is finding ways to test them. The ever-growing understanding of human genetics is revealing connections between peoples that have never been seen before.
Super Bowl XXXIV Footballs and 2000 Summer Olympic SouvenirsThe NFL used DNA technology to tag all the Super Bowl XXXIV balls, ensuring their authenticity for years to come and helping to combat the growing epidemic of sports memorabilia fraud. The footballs were marked with an invisible, yet permanent, strand of synthetic DNA. The DNA strand is unique and is verifiable any time in the future using a specially calibrated laser.
A section of human genetic code taken from several unnamed Australian athletes was added to ink used to mark all official goods — everything from caps to socks — from the 2000 Summer Olympic Games. The technology is used as a way to mark artwork or one-of-a-kind sports souvenirs.
Migration PatternsEvolutionarily stable mitochondrial DNA and Y chromosomes have allowed bioanthropologists to begin to trace human migration patterns around the world and identify family lineage
See Genetic Anthropology, Ancestry, and Ancient Human Migrations
Wine Heritage Using DNA fingerprinting techniques akin to those used to solve crimes and settle paternity suits, scientists at the University of California, Davis, have discovered that 18 of the world's most renowned grapevine varieties, or cultivars are close relatives. These include varieties long grown in northeastern France such as Chardonnay, the "king of whites," and reds such as Pinot and Gamay noir, are close relatives.
DNA Banks for Endangered Animal Species
Poached Animals
Declining Grizzly Bear Population
Snowball the Cat A woman was murdered in Prince Edward Island, Canada. Her estranged husband was implicated because a snowy white cat hair was found in a jacket near the scene of the crime, and DNA fragments from the hair matched DNA fragments from Snowball, the cat belonging to the husband's parents. See M. Menotti-Raymond et al., "Pet cat hair implicates murder suspect," Nature, 386, 774, 1997. Also see Holmes, Judy, Feline Forensics, Syracuse University Magazine, Summer 2001.
Angiosperm Witness for the Prosecution The first case in which a murderer was convicted on plant DNA evidence was described in the PBS TV series, "Scientific American Frontiers." A young woman was murdered in Phoenix, Arizona, and a pager found at the scene of the crime led the police to a prime suspect. He admitted picking up the victim but claimed she had robbed him of his wallet and pager. The forensic squad examined the suspect's pickup truck and collected pods later identified as the fruits of the palo verde tree (Cercidium spp.). One detective went back to the murder scene and found several Palo Verde trees, one of which showed damage that could have been caused by a vehicle. The detective's superior officer innocently suggested the possibility of linking the fruits and the tree by using DNA comparison, not realizing that this had never been done before. Several researchers were contacted before a geneticist at the University of Arizona in Tucson agreed to take on the case. Of course, it was crucial to establish evidence that would stand up in court on whether individual plants (especially Palo Verde trees) have unique patterns of DNA. A preliminary study on samples from different trees at the murder scene and elsewhere quickly established that each Palo Verde tree is unique in its DNA pattern. It was then a simple matter to link the pods from the suspect's truck to the damaged tree at the murder scene and obtain a conviction. [WNED-TV (PBS - Buffalo, N.Y.)]
May 9, 2008
DNA Forensics - DNA Fingerpring
How does forensic identification work?
Any type of organism can be identified by examination of DNA sequences unique to that species. Identifying individuals within a species is less precise at this time, although when DNA sequencing technologies progress farther, direct comparison of very large DNA segments, and possibly even whole genomes, will become feasible and practical and will allow precise individual identification.
To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of regions.
Some Examples of DNA Uses for Forensic Identification
Identify potential suspects whose DNA may match evidence left at crime scenes
Exonerate persons wrongly accused of crimes
Identify crime and catastrophe victims
Establish paternity and other family relationships
Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers)
Detect bacteria and other organisms that may pollute air, water, soil, and food
Match organ donors with recipients in transplant programs
Determine pedigree for seed or livestock breeds
Authenticate consumables such as caviar and wine
Is DNA effective in identifying persons?
[answer provided by Daniel Drell of the U.S. DOE Human Genome Program]
DNA identification can be quite effective if used intelligently. Portions of the DNA sequence that vary the most among humans must be used; also, portions must be large enough to overcome the fact that human mating is not absolutely random.
Consider the scenario of a crime scene investigation . . .
Assume that type O blood is found at the crime scene. Type O occurs in about 45% of Americans. If investigators type only for ABO, finding that the "suspect" in a crime is type O really doesn't reveal very much.
If, in addition to being type O, the suspect is a blond, and blond hair is found at the crime scene, you now have two bits of evidence to suggest who really did it. However, there are a lot of Type O blonds out there.
If you find that the crime scene has footprints from a pair of Nike Air Jordans (with a distinctive tread design) and the suspect, in addition to being type O and blond, is also wearing Air Jordans with the same tread design, you are much closer to linking the suspect with the crime scene.
In this way, by accumulating bits of linking evidence in a chain, where each bit by itself isn't very strong but the set of all of them together is very strong, you can argue that your suspect really is the right person.
With DNA, the same kind of thinking is used; you can look for matches (based on sequence or on numbers of small repeating units of DNA sequence) at many different locations on the person's genome; one or two (even three) aren't enough to be confident that the suspect is the right one, but four (sometimes five) are used. A match at all five is rare enough that you (or a prosecutor or a jury) can be very confident ("beyond a reasonable doubt") that the right person is accused.
How is DNA typing done?
Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products.
In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions (markers).
Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. A marker by itself usually is not unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person.
If the sample profiles don't match, the person did not contribute the DNA at the crime scene.
If the patterns match, the suspect may have contributed the evidence sample. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim. The question is, How small do the odds have to be when conviction of the guilty or acquittal of the innocent lies in the balance? Many judges consider this a matter for a jury to take into consideration along with other evidence in the case. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are about 50:50.
The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. Four to six probes are recommended. Testing with several more probes will become routine, observed John Hicks (Alabama State Department of Forensic Services). He predicted that DNA chip technology (in which thousands of short DNA sequences are embedded in a tiny chip) will enable much more rapid, inexpensive analyses using many more probes and raising the odds against coincidental matches.
What are some of the DNA technologies used in forensic investigations?
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample.
RFLP was one of the first applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA-analysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with RFLP.
PCR Analysis
Polymerase chain reaction (PCR) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.
STR Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in a billion.
Mitochondrial DNA Analysis
Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable.
All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing-person investigations.
Y-Chromosome Analysis
The Y chromosome is passed directly from father to son, so analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors.
Any type of organism can be identified by examination of DNA sequences unique to that species. Identifying individuals within a species is less precise at this time, although when DNA sequencing technologies progress farther, direct comparison of very large DNA segments, and possibly even whole genomes, will become feasible and practical and will allow precise individual identification.
To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of regions.
Some Examples of DNA Uses for Forensic Identification
Identify potential suspects whose DNA may match evidence left at crime scenes
Exonerate persons wrongly accused of crimes
Identify crime and catastrophe victims
Establish paternity and other family relationships
Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers)
Detect bacteria and other organisms that may pollute air, water, soil, and food
Match organ donors with recipients in transplant programs
Determine pedigree for seed or livestock breeds
Authenticate consumables such as caviar and wine
Is DNA effective in identifying persons?
[answer provided by Daniel Drell of the U.S. DOE Human Genome Program]
DNA identification can be quite effective if used intelligently. Portions of the DNA sequence that vary the most among humans must be used; also, portions must be large enough to overcome the fact that human mating is not absolutely random.
Consider the scenario of a crime scene investigation . . .
Assume that type O blood is found at the crime scene. Type O occurs in about 45% of Americans. If investigators type only for ABO, finding that the "suspect" in a crime is type O really doesn't reveal very much.
If, in addition to being type O, the suspect is a blond, and blond hair is found at the crime scene, you now have two bits of evidence to suggest who really did it. However, there are a lot of Type O blonds out there.
If you find that the crime scene has footprints from a pair of Nike Air Jordans (with a distinctive tread design) and the suspect, in addition to being type O and blond, is also wearing Air Jordans with the same tread design, you are much closer to linking the suspect with the crime scene.
In this way, by accumulating bits of linking evidence in a chain, where each bit by itself isn't very strong but the set of all of them together is very strong, you can argue that your suspect really is the right person.
With DNA, the same kind of thinking is used; you can look for matches (based on sequence or on numbers of small repeating units of DNA sequence) at many different locations on the person's genome; one or two (even three) aren't enough to be confident that the suspect is the right one, but four (sometimes five) are used. A match at all five is rare enough that you (or a prosecutor or a jury) can be very confident ("beyond a reasonable doubt") that the right person is accused.
How is DNA typing done?
Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products.
In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions (markers).
Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. A marker by itself usually is not unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person.
If the sample profiles don't match, the person did not contribute the DNA at the crime scene.
If the patterns match, the suspect may have contributed the evidence sample. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim. The question is, How small do the odds have to be when conviction of the guilty or acquittal of the innocent lies in the balance? Many judges consider this a matter for a jury to take into consideration along with other evidence in the case. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are about 50:50.
The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. Four to six probes are recommended. Testing with several more probes will become routine, observed John Hicks (Alabama State Department of Forensic Services). He predicted that DNA chip technology (in which thousands of short DNA sequences are embedded in a tiny chip) will enable much more rapid, inexpensive analyses using many more probes and raising the odds against coincidental matches.
What are some of the DNA technologies used in forensic investigations?
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample.
RFLP was one of the first applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA-analysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with RFLP.
PCR Analysis
Polymerase chain reaction (PCR) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.
STR Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in a billion.
Mitochondrial DNA Analysis
Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable.
All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing-person investigations.
Y-Chromosome Analysis
The Y chromosome is passed directly from father to son, so analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors.
DNA in Cell
1 . Epigenome Sequencing Comes of Age
Jian-Kang Zhu Cell 133: 395-397.[Full Text] [PDF]
Epigenetic states are responsive to developmental and environmental signals, and as a consequence a eukaryotic cell can have many different epigenomes. In this issue of Cell, Lister et al., 2008Chan et al., 2006Collart et al., Danilova et al., 2007Nudler et al., 2002Bernstein et al., 2007Gnatt et al., 2001Lee et al., 2005Bicknell et al., 2004Brenner, 1974Bishop et al., 1992Bernstein et al., 2005Blat et al., 1999Dyda et al., 1994Cheung et al., 2000Burley et al., 1985Chen et al., 2005Benard et al., 2001Ebersole et al., 2000 present the floral epigenome of Arabidopsis using next-generation sequencing technology to analyze both DNA methylation at single-base resolution and the expression of small RNAs.
2 . Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis
Ryan Lister, Ronan C. O'Malley, Julian Tonti-Filippini, Brian D. Gregory, Charles C. Berry, A. Harvey Millar and Joseph R. Ecker Cell : .[Full Text] [PDF]
Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.
3 . MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks
Manuel Stucki, Julie A. Clapperton, Duaa Mohammad, Michael B. Yaffe, Stephen J. Smerdon and Stephen P. Jackson Cell 133: 549.[Full Text] [PDF]
4 . Meiosis I Is Established through Division-Specific Translational Control of a Cyclin
Thomas M. Carlile and Angelika Amon Cell 133: 280-291.[Full Text] [PDF]
In budding yeast, key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II, through 5′UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.
5 . Independent Positioning and Action of Escherichia coli Replisomes in Live Cells
Rodrigo Reyes-Lamothe, Christophe Possoz, Olessia Danilova and David J. Sherratt Cell 133: 90-102.[Full Text] [PDF]
A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.
6 . Transcription Termination: Pulling Out All the Stops
Jack F. Greenblatt Cell 132: 917-918.[Full Text] [PDF]
In this issue, Huettel et al., 2006Cao et al., 2002Cohen-Fix et al., 1996Cook, 1999Larson et al., 2008Bernstein et al., 2006Davenport et al., 2000Hunter, Berger et al., Booth et al., 2001Bailis et al., 1998Bernstein et al., 2004Birney et al., 2007DeLano, 2002Caretti et al., 2003Bunting et al., 2003Chartier-Harlin et al., 2004Bakkenist et al., 2004Earnshaw et al., 1989 describe the use of optical traps to pull on the DNA template or RNA transcript and thereby explore the termination mechanism for E. coli RNA polymerase at intrinsic terminators. Their results imply that, depending on the nature of the terminator sequence, RNA polymerase uses either hypertranslocation or RNA:DNA shearing to destabilize the hybrid in the transcription bubble.
7 . FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription
Mathieu Lupien, Jérôme Eeckhoute, Clifford A. Meyer, Qianben Wang, Yong Zhang, Wei Li, Jason S. Carroll, X. Shirley Liu and Myles Brown Cell 132: 958-970.[Full Text] [PDF]
Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.
8 . Applied Force Reveals Mechanistic and Energetic Details of Transcription Termination
Matthew H. Larson, William J. Greenleaf, Robert Landick and Steven M. Block Cell 132: 971-982.[Full Text] [PDF]
Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2–3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.
9 . Hop1 and the Meiotic DNA-Damage Response
Neil Hunter Cell 132: 731-732.[Full Text] [PDF]
During the DNA-damage response, adaptor proteins mediate signaling between the PI3K-like sensor kinases, ATM and ATR, and serine/threonine effector kinases. Cokus et al., 2008Bernstein et al., 2007Bloom et al., 2007Bates et al., 2005Gusarov et al., 1999Badve et al., 2007Adelman et al., 2002Carballo et al., 2008Agius et al., 2006Anderson et al., 2002Anuradha et al., 2004Albert et al., 2007Ansel et al., 2003Brünger et al., 1998Brown et al., 2003Argiriadi et al., 2006Blanco-Rodríguez, 2002Ahmed et al., 2001Chan et al., 2005 now show that the chromosomal protein Hop1 mediates PI3K-like kinase signaling during the repair of DNA double-strand breaks (DSBs) in meiosis.
10 . Cellular Programming of Plant Gene Imprinting
Jin Hoe Huh, Matthew J. Bauer, Tzung-Fu Hsieh and Robert L. Fischer Cell 132: 735-744.[Full Text] [PDF]
Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm.
11 . C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins
Marcela Raices, Ramiro E. Verdun, Sarah A. Compton, Candy I. Haggblom, Jack D. Griffith, Andrew Dillin and Jan Karlseder Cell 132: 745-757.[Full Text] [PDF]
Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3′ G-strand overhangs as well as 5′ C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5′ and 3′ single-stranded termini.
12 . Phosphorylation of the Axial Element Protein Hop1 by Mec1/Tel1 Ensures Meiotic Interhomolog Recombination
Jesús A. Carballo, Anthony L. Johnson, Steven G. Sedgwick and Rita S. Cha Cell 132: 758-770.[Full Text] [PDF]
An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.
13 . Dynamic Regulation of Nucleosome Positioning in the Human Genome
Dustin E. Schones, Kairong Cui, Suresh Cuddapah, Tae-Young Roh, Artem Barski, Zhibin Wang, Gang Wei and Keji Zhao Cell 132: 887-898.[Full Text] [PDF]
The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However, nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. We find that nucleosome phasing relative to the transcription start sites is directly correlated to RNA polymerase II (Pol II) binding. Furthermore, the first nucleosome downstream of a start site exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the −1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome.
14 . Cohesins Functionally Associate with CTCF on Mammalian Chromosome Arms
Vania Parelho, Suzana Hadjur, Mikhail Spivakov, Marion Leleu, Stephan Sauer, Heather C. Gregson, Adam Jarmuz, Claudia Canzonetta, Zoe Webster, Tatyana Nesterova, Bradley S. Cobb, Kyoko Yokomori, Niall Dillon, Luis Aragon, Amanda G. Fisher and Matthias Merkenschlager Cell 132: 422-433.[Full Text] [PDF]
Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.
15 . Mechanism of IS200/IS605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection
Orsolya Barabas, Donald R. Ronning, Catherine Guynet, Alison Burgess Hickman, Bao Ton-Hoang, Michael Chandler and Fred Dyda Cell 132: 208-220.[Full Text] [PDF]
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
16 . Chk1 Is a Histone H3 Threonine 11 Kinase that Regulates DNA Damage-Induced Transcriptional Repression
Midori Shimada, Hiroyuki Niida, Doaa H. Zineldeen, Hideaki Tagami, Masafumi Tanaka, Hiroyuki Saito and Makoto Nakanishi Cell 132: 221-232.[Full Text] [PDF]
DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.
17 . Structure of a Sliding Clamp on DNA
Roxana E. Georgescu, Seung-Sup Kim, Olga Yurieva, John Kuriyan, Xiang-Peng Kong and Mike O'Donnell Cell 132: 43-54.[Full Text] [PDF]
The structure of the E. coli β clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the β ring. The pronounced 22° angle of DNA through β may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.
18 . A DNA Replication Mechanism for Generating Nonrecurrent Rearrangements Associated with Genomic Disorders
Jennifer A. Lee, Claudia M.B. Carvalho and James R. Lupski Cell 131: 1235-1247.[Full Text] [PDF]
The prevailing mechanism for recurrent and some nonrecurrent rearrangements causing genomic disorders is nonallelic homologous recombination (NAHR) between region-specific low-copy repeats (LCRs). For other nonrecurrent rearrangements, nonhomologous end joining (NHEJ) is implicated. Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused most frequently (60%–70%) by nonrecurrent duplication of the dosage-sensitive proteolipid protein 1 (PLP1) gene but also by nonrecurrent deletion or point mutations. Many PLP1 duplication junctions are refractory to breakpoint sequence analysis, an observation inconsistent with a simple recombination mechanism. Our current analysis of junction sequences in PMD patients confirms the occurrence of simple tandem PLP1 duplications but also uncovers evidence for sequence complexity at some junctions. These data are consistent with a replication-based mechanism that we term FoSTeS, for replication Fork Stalling and Template Switching. We propose that complex duplication and deletion rearrangements associated with PMD, and potentially other nonrecurrent rearrangements, may be explained by this replication-based mechanism.
19 . Tel2 Regulates the Stability of PI3K-Related Protein Kinases
Hiroyuki Takai, Richard C. Wang, Kaori K. Takai, Haijuan Yang and Titia de Lange Cell 131: 1248-1259.[Full Text] [PDF]
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
20 . CENP-B Controls Centromere Formation Depending on the Chromatin Context
Teruaki Okada, Jun-ichirou Ohzeki, Megumi Nakano, Kinya Yoda, William R. Brinkley, Vladimir Larionov and Hiroshi Masumoto Cell 131: 1287-1300.[Full Text] [PDF]
The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.
Jian-Kang Zhu Cell 133: 395-397.[Full Text] [PDF]
Epigenetic states are responsive to developmental and environmental signals, and as a consequence a eukaryotic cell can have many different epigenomes. In this issue of Cell, Lister et al., 2008Chan et al., 2006Collart et al., Danilova et al., 2007Nudler et al., 2002Bernstein et al., 2007Gnatt et al., 2001Lee et al., 2005Bicknell et al., 2004Brenner, 1974Bishop et al., 1992Bernstein et al., 2005Blat et al., 1999Dyda et al., 1994Cheung et al., 2000Burley et al., 1985Chen et al., 2005Benard et al., 2001Ebersole et al., 2000 present the floral epigenome of Arabidopsis using next-generation sequencing technology to analyze both DNA methylation at single-base resolution and the expression of small RNAs.
2 . Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis
Ryan Lister, Ronan C. O'Malley, Julian Tonti-Filippini, Brian D. Gregory, Charles C. Berry, A. Harvey Millar and Joseph R. Ecker Cell : .[Full Text] [PDF]
Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.
3 . MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks
Manuel Stucki, Julie A. Clapperton, Duaa Mohammad, Michael B. Yaffe, Stephen J. Smerdon and Stephen P. Jackson Cell 133: 549.[Full Text] [PDF]
4 . Meiosis I Is Established through Division-Specific Translational Control of a Cyclin
Thomas M. Carlile and Angelika Amon Cell 133: 280-291.[Full Text] [PDF]
In budding yeast, key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II, through 5′UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.
5 . Independent Positioning and Action of Escherichia coli Replisomes in Live Cells
Rodrigo Reyes-Lamothe, Christophe Possoz, Olessia Danilova and David J. Sherratt Cell 133: 90-102.[Full Text] [PDF]
A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.
6 . Transcription Termination: Pulling Out All the Stops
Jack F. Greenblatt Cell 132: 917-918.[Full Text] [PDF]
In this issue, Huettel et al., 2006Cao et al., 2002Cohen-Fix et al., 1996Cook, 1999Larson et al., 2008Bernstein et al., 2006Davenport et al., 2000Hunter, Berger et al., Booth et al., 2001Bailis et al., 1998Bernstein et al., 2004Birney et al., 2007DeLano, 2002Caretti et al., 2003Bunting et al., 2003Chartier-Harlin et al., 2004Bakkenist et al., 2004Earnshaw et al., 1989 describe the use of optical traps to pull on the DNA template or RNA transcript and thereby explore the termination mechanism for E. coli RNA polymerase at intrinsic terminators. Their results imply that, depending on the nature of the terminator sequence, RNA polymerase uses either hypertranslocation or RNA:DNA shearing to destabilize the hybrid in the transcription bubble.
7 . FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription
Mathieu Lupien, Jérôme Eeckhoute, Clifford A. Meyer, Qianben Wang, Yong Zhang, Wei Li, Jason S. Carroll, X. Shirley Liu and Myles Brown Cell 132: 958-970.[Full Text] [PDF]
Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.
8 . Applied Force Reveals Mechanistic and Energetic Details of Transcription Termination
Matthew H. Larson, William J. Greenleaf, Robert Landick and Steven M. Block Cell 132: 971-982.[Full Text] [PDF]
Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2–3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.
9 . Hop1 and the Meiotic DNA-Damage Response
Neil Hunter Cell 132: 731-732.[Full Text] [PDF]
During the DNA-damage response, adaptor proteins mediate signaling between the PI3K-like sensor kinases, ATM and ATR, and serine/threonine effector kinases. Cokus et al., 2008Bernstein et al., 2007Bloom et al., 2007Bates et al., 2005Gusarov et al., 1999Badve et al., 2007Adelman et al., 2002Carballo et al., 2008Agius et al., 2006Anderson et al., 2002Anuradha et al., 2004Albert et al., 2007Ansel et al., 2003Brünger et al., 1998Brown et al., 2003Argiriadi et al., 2006Blanco-Rodríguez, 2002Ahmed et al., 2001Chan et al., 2005 now show that the chromosomal protein Hop1 mediates PI3K-like kinase signaling during the repair of DNA double-strand breaks (DSBs) in meiosis.
10 . Cellular Programming of Plant Gene Imprinting
Jin Hoe Huh, Matthew J. Bauer, Tzung-Fu Hsieh and Robert L. Fischer Cell 132: 735-744.[Full Text] [PDF]
Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm.
11 . C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins
Marcela Raices, Ramiro E. Verdun, Sarah A. Compton, Candy I. Haggblom, Jack D. Griffith, Andrew Dillin and Jan Karlseder Cell 132: 745-757.[Full Text] [PDF]
Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3′ G-strand overhangs as well as 5′ C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5′ and 3′ single-stranded termini.
12 . Phosphorylation of the Axial Element Protein Hop1 by Mec1/Tel1 Ensures Meiotic Interhomolog Recombination
Jesús A. Carballo, Anthony L. Johnson, Steven G. Sedgwick and Rita S. Cha Cell 132: 758-770.[Full Text] [PDF]
An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.
13 . Dynamic Regulation of Nucleosome Positioning in the Human Genome
Dustin E. Schones, Kairong Cui, Suresh Cuddapah, Tae-Young Roh, Artem Barski, Zhibin Wang, Gang Wei and Keji Zhao Cell 132: 887-898.[Full Text] [PDF]
The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However, nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. We find that nucleosome phasing relative to the transcription start sites is directly correlated to RNA polymerase II (Pol II) binding. Furthermore, the first nucleosome downstream of a start site exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the −1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome.
14 . Cohesins Functionally Associate with CTCF on Mammalian Chromosome Arms
Vania Parelho, Suzana Hadjur, Mikhail Spivakov, Marion Leleu, Stephan Sauer, Heather C. Gregson, Adam Jarmuz, Claudia Canzonetta, Zoe Webster, Tatyana Nesterova, Bradley S. Cobb, Kyoko Yokomori, Niall Dillon, Luis Aragon, Amanda G. Fisher and Matthias Merkenschlager Cell 132: 422-433.[Full Text] [PDF]
Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.
15 . Mechanism of IS200/IS605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection
Orsolya Barabas, Donald R. Ronning, Catherine Guynet, Alison Burgess Hickman, Bao Ton-Hoang, Michael Chandler and Fred Dyda Cell 132: 208-220.[Full Text] [PDF]
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
16 . Chk1 Is a Histone H3 Threonine 11 Kinase that Regulates DNA Damage-Induced Transcriptional Repression
Midori Shimada, Hiroyuki Niida, Doaa H. Zineldeen, Hideaki Tagami, Masafumi Tanaka, Hiroyuki Saito and Makoto Nakanishi Cell 132: 221-232.[Full Text] [PDF]
DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.
17 . Structure of a Sliding Clamp on DNA
Roxana E. Georgescu, Seung-Sup Kim, Olga Yurieva, John Kuriyan, Xiang-Peng Kong and Mike O'Donnell Cell 132: 43-54.[Full Text] [PDF]
The structure of the E. coli β clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the β ring. The pronounced 22° angle of DNA through β may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.
18 . A DNA Replication Mechanism for Generating Nonrecurrent Rearrangements Associated with Genomic Disorders
Jennifer A. Lee, Claudia M.B. Carvalho and James R. Lupski Cell 131: 1235-1247.[Full Text] [PDF]
The prevailing mechanism for recurrent and some nonrecurrent rearrangements causing genomic disorders is nonallelic homologous recombination (NAHR) between region-specific low-copy repeats (LCRs). For other nonrecurrent rearrangements, nonhomologous end joining (NHEJ) is implicated. Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused most frequently (60%–70%) by nonrecurrent duplication of the dosage-sensitive proteolipid protein 1 (PLP1) gene but also by nonrecurrent deletion or point mutations. Many PLP1 duplication junctions are refractory to breakpoint sequence analysis, an observation inconsistent with a simple recombination mechanism. Our current analysis of junction sequences in PMD patients confirms the occurrence of simple tandem PLP1 duplications but also uncovers evidence for sequence complexity at some junctions. These data are consistent with a replication-based mechanism that we term FoSTeS, for replication Fork Stalling and Template Switching. We propose that complex duplication and deletion rearrangements associated with PMD, and potentially other nonrecurrent rearrangements, may be explained by this replication-based mechanism.
19 . Tel2 Regulates the Stability of PI3K-Related Protein Kinases
Hiroyuki Takai, Richard C. Wang, Kaori K. Takai, Haijuan Yang and Titia de Lange Cell 131: 1248-1259.[Full Text] [PDF]
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
20 . CENP-B Controls Centromere Formation Depending on the Chromatin Context
Teruaki Okada, Jun-ichirou Ohzeki, Megumi Nakano, Kinya Yoda, William R. Brinkley, Vladimir Larionov and Hiroshi Masumoto Cell 131: 1287-1300.[Full Text] [PDF]
The centromere is a chromatin region that serves as the spindle attachment point and directs accurate inheritance of eukaryotic chromosomes during cell divisions. However, the mechanism by which the centromere assembles and stabilizes at a specific genomic region is not clear. The de novo formation of a human/mammalian artificial chromosome (HAC/MAC) with a functional centromere assembly requires the presence of alpha-satellite DNA containing binding motifs for the centromeric CENP-B protein. We demonstrate here that de novo centromere assembly on HAC/MAC is dependent on CENP-B. In contrast, centromere formation is suppressed in cells expressing CENP-B when alpha-satellite DNA was integrated into a chromosomal site. Remarkably, on those integration sites CENP-B enhances histone H3-K9 trimethylation and DNA methylation, thereby stimulating heterochromatin formation. Thus, we propose that CENP-B plays a dual role in centromere formation, ensuring de novo formation on DNA lacking a functional centromere but preventing the formation of excess centromeres on chromosomes.
DNA in Nature
1. A role for DNA primase in coupling DNA replication to DNA damage response
Federica Marini, Achille Pellicioli, Vera Paciotti, Giovanna Lucchini, Paolo Plevani, David F. Stern, Marco Foiani
SUMMARY: The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging
CONTEXT: Introduction Eukaryotic cells have developed a network of highly conserved surveillance mechanisms (checkpoints), ensuring that damaged chromosomes are repaired before being replicated or segregated. These mechanisms are essential for...
The EMBO Journal 16, 639 - 650 (01 Feb 1997), doi: 10.1093/emboj/16.3.639, Article
2. Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks
Dale A. Ramsden, Martin Gellert
SUMMARY: Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand
CONTEXT: Introduction DNA double-strand breaks (DSBs) result from a variety of exogenous DNA-damaging agents, such as ionizing radiation. They are repaired either by using an intact copy of the broken region as a template (homologous...
The EMBO Journal 17, 609 - 614 (15 Jan 1998), doi: 10.1093/emboj/17.2.609, Article
3. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes
Rong-Guang Shao, Chun-Xia Cao, Hongliang Zhang, Kurt W. Kohn, Marc S. Wold, Yves Pommier
SUMMARY: Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase
CONTEXT: Introduction Replication protein A (RPA) is an ubiquitous eukaryotic single-stranded DNA (ssDNA)-binding protein complex, which was originally identified as an essential factor for simian virus 40 (SV40) DNA replication in vitro (Wobbe...
The EMBO Journal 18, 1397 - 1406 (01 Mar 1999), doi: 10.1093/emboj/18.5.1397, Article
4. Human DNA polymerase β initiates DNA synthesis during long-patch repair of reduced AP sites in DNA
Andrej Ja. Podlutsky, Irina I. Dianova, Vladimir N. Podust, Vilhelm A. Bohr, Grigory L. Dianov
SUMMARY: Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase β (Pol
CONTEXT: Introduction Abasic (apurinic/apyrimidinic, AP) sites can arise in DNA as a result of spontaneous hydrolysis of the N-glycosylic bond or the removal of altered bases by DNA glycosylases (Lindahl, 1993). It has been estimated that 10 000...
The EMBO Journal 20, 1477 - 1482 (15 Mar 2001), doi: 10.1093/emboj/20.6.1477, Article
5. MOT1-catalyzed TBP–DNA disruption: uncoupling DNA conformational change and role of upstream DNA
Russell P. Darst, Dongyan Wang, David T. Auble
SUMMARY: SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein–DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood. Here we examine the
CONTEXT: Introduction The ATPase activities of SNF2/SWI2-related proteins are of at least two types (Pazin and Kadonaga, 1997; Vignali et al., 2000). Many proteins in this family possess DNA-stimulated ATPase activity. ATPases with DNA-stimulated...
The EMBO Journal 20, 2028 - 2040 (17 Apr 2001), doi: 10.1093/emboj/20.8.2028, Article
6. Crystal structure of the CENP-B protein–DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA
Yoshinori Tanaka, Osamu Nureki, Hitoshi Kurumizaka, Shuya Fukai, Shinichi Kawaguchi, Mari Ikuta, Junji Iwahara, Tsuneko Okazaki, Shigeyuki Yokoyama
SUMMARY: The human centromere protein B (CENP-B), one of the centromere components, specifically binds a 17 bp sequence (the CENP-B box), which appears in every
CONTEXT: Introduction The centromere is a region of the chromosome essential for its segregation during cell division, and has a special chromatin structure involving -satellite DNA repeats and their associated proteins. In the human, the...
The EMBO Journal 20, 6612 - 6618 (03 Dec 2001), doi: 10.1093/emboj/20.23.6612, Article
7. Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs
Jasminka Boskovic, Angel Rivera-Calzada, Joseph D. Maman, Pablo Chacón, Keith R. Willison, Laurence H. Pearl, Oscar Llorca
SUMMARY: The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is essential for the repair of double-stranded DNA breaks (DSBs) in non- homologous end joining
CONTEXT: Introduction DNA is constantly being exposed to insults that compromise its integrity and its information content. Ionizing radiation and some chemotherapeutic drugs generate double-stranded DNA breaks (DSBs) that must be repaired to...
The EMBO Journal 22, 5875 - 5882 (03 Nov 2003), doi: 10.1093/emboj/cdg555, Article
8. Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV
Christine Anne Koch, Roger Agyei, Sarah Galicia, Pavel Metalnikov, Paul O'Donnell, Andrei Starostine, Michael Weinfeld, Daniel Durocher
SUMMARY: Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA
CONTEXT: Introduction DNA double-strand breaks (DSBs) pose a major threat to cell survival and genome stability. If left unrepaired, DSBs can result in the loss of genetic material or cell death. Moreover, mutations or gross genetic aberrations...
The EMBO Journal 23, 3874 - 3885 (29 Sep 2004), doi: 10.1038/sj.emboj.7600375, Article
9. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC–DNA binding
Dirk Remus, Eileen L Beall, Michael R Botchan
SUMMARY: Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However,
CONTEXT: Introduction In a eukaryotic cell committed to duplication, chromosomal DNA replication initiates at many sites called origins of DNA replication. The process that determines origin selection is understood in some depth for fungal...
The EMBO Journal 23, 897 - 907 (25 Feb 2004), doi: 10.1038/sj.emboj.7600077, Article
10. p53 is involved in regulation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) by DNA damaging agents
Thomas Grombacher, Uta Eichhorn, Bernd Kaina
SUMMARY: The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is inducible by genotoxic stress. MGMT induction results from transcriptional activation of the MGMT gene which is
CONTEXT: C:\WINDOWS>echo off
Oncogene 17, 845 - 851 (18 Aug 1998), doi: 10.1038/sj.onc.1202000, Original Article
11. Structure of the Elk-1–DNA complex reveals how DNA-distal residues affect ETS domain recognition of DNA
SUMMARY: SAP-1 and Elk-1 are members of a large group of eukaryotic transcription factors that contain a conserved ETS DNA binding domain and that cooperate
CONTEXT: The ETS (E26 transformation specific or E twenty-six) domain transcription factors form a family of eukaryotic proteins with highly conserved DNA binding domains and as such are used as a paradigm for studying DNA binding specificity....
Nature Structural Biology 7, 292 - 297 (01 Apr 2000), doi: 10.1038/74055, Letters
12. A DNA-dependent DNA Polymerase and a DNA Endonuclease in Virions of Rous Sarcoma Virus
SATOSHI MIZUTANI, DAVID BOETTIGER, HOWARD M. TEMIN
SUMMARY: The virion contains a DNA-dependent DNA polymerase which replicates duplex DNA and an endonuclease which attacks it. A ligase system may also be present.
CONTEXT: The virion contains a DNA-dependent DNA polymerase which replicates duplex DNA and an endonuclease which attacks it. A ligase system may also be present. THE replication of RNA tumour viruses seems to involve a DNA intermediate-the DNA...
Nature 228, 424 - 427 (31 Oct 1970), doi: 10.1038/228424a0, Article
13. DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination
Clifford D. Mol, Tadahide Izumi, Sankar Mitra, John A. Tainer
SUMMARY: Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repa
CONTEXT: Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA...
Nature 403, 451 - 456 (27 Jan 2000), doi: 10.1038/35000249, Letter
14. Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends
Kristina Staley, Anne J Blaschke, Jerold Chun
SUMMARY: Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal `ladder'. However, nucleosomal
CONTEXT: C:\WINDOWS>echo off
Cell Death and Differentiation 4, 66 - 75 (04 Dec 1996), doi: 10.1038/sj.cdd.4400207, Original Article
15. Genetic interaction between DNA polymerase β and DNA-PKcs in embryogenesis and neurogenesis
N Niimi,
SUMMARY: DNA polymerase β (Polβ) has been implicated in base excision repair. Polβ knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth.
CONTEXT: Introduction The genome is continuously damaged by a variety of endogenous and exogenous agents. Repair of such damage is a crucial mechanism for maintaining genomic integrity. A failure in faithful repair causes mutations with an...
Cell Death and Differentiation 12, 184 - 191 (01 Feb 2005), doi: 10.1038/sj.cdd.4401543, Original Paper
16. Retroviral DNA integration and the DNA damage response
A M Skalka, R A Katz
SUMMARY: Retroviral DNA integration creates a discontinuity in the host cell chromatin and repair of this damage is required to complete the integration process. As
CONTEXT: Introduction Viruses are obligate parasites that require host cell functions for their survival and propagation. Successful viruses have evolved mechanisms to exploit and/or counteract host defense systems, and modify or redirect normal...
Cell Death and Differentiation 12, 971 - 978 (01 Aug 2005), doi: 10.1038/sj.cdd.4401573, Reviews
17. DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice
SUMMARY: The oncofetal alpha-fetoprotein (AFP) is reexpressed in the majority of hepatocellular carcinomas and may be used as a target molecule for an immunotherapy or
CONTEXT: Hepatocellular carcinoma (HCC) is a common malignancy with a very poor prognosis. Only a minority of patients are eligible for surgical therapies due to advanced tumors or extrahepatic disease at primary diagnosis.1,2 Therefore, novel...
Cancer Gene Therapy 9, 346 - 355 (03 Apr 2002), doi: 10.1038/sj.cgt.7700445, Original Article
18. A novel protein-DNA interaction involved with the CpG dinucleotide at -30 upstream is linked to the DNA methylation mediated transcription silencing of the MAGE-A1 gene
Jie ZHANG, Jian YU, Jun GU, Bao Mei GAO, Ying Jun ZHAO, Peng WANG, Hong Yu ZHANG, Jing De ZHU
SUMMARY: ABSTRACT To understand the DNA-methylation mediated gene silencing mechanisms, we analyzed in cell culture of the promoter function of the MAGE-A1 gene, which is frequently
CONTEXT: INTRODUCTION Addition of a methyl group at the fifth carbon of the cytosine in the CpG dinucleotide is the sole covalent modification of DNA in vertebrate genome and recognized as an important epigenetic signature 1. The vital importance...
Cell Research 14, 283 - 294 (00 Aug 2004), doi: 10.1038/sj.cr.7290229, Article
19. LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts
Zhi Gang LU, Chuan Mao ZHANG, Zhong He ZHAI
SUMMARY: ABSTRACT DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation
CONTEXT: INTRODUCTION Apoptosis, a morphologically distinct form of programmed cell death, has attracted considerable attention for its implicated roles in embryonic development, tissue homeostasis and protection against diseases. A cell in...
Cell Research 14, 134 - 140 (00 Apr 2004), doi: 10.1038/sj.cr.7290212, Article
20. Homologous recombination in DNA repair and DNA damage tolerance
Xuan Li, Wolf-Dietrich Heyer
SUMMARY: Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombi
CONTEXT: Introduction DNA damage is a fact of life as a consequence of endogenous sources and processes as well as exogenous sources 1. Homologous recombination (HR) is a DNA metabolic process found in all forms of life that provides...
Cell Research 18, 99 - 113 (07 Jan 2008), doi: 10.1038/cr.2008.1, Review
21. XRCC1 and DNA polymerase [beta] in cellular protection against cytotoxic DNA single-strand breaks
Julie K Horton, Mary Watson, Donna F Stefanick, Daniel T Shaughnessy, Jack A Taylor, Samuel H Wilson
SUMMARY: Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5|[
CONTEXT: Introduction Cells have evolved intricate DNA repair mechanisms to circumvent genomic instability. Thousands of spontaneous single-strand breaks (SSBs) occur in cellular DNA each day 1 and, if they persist, can convert to potentially...
Cell Research 18, 48 - 63 (07 Jan 2008), doi: 10.1038/cr.2008.7, Review
22. Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair
Longhou Fang, YiGuo Wang, Dan Du, Guang Yang, Tim Tak Kwok, Siu Kai Kong, Benjamin Chen, David J Chen, Zhengjun Chen
SUMMARY: The partitioning-defective 3 (Par3), a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of
CONTEXT: Introduction Cell polarity is vital for the development of multicellular organisms and for the proper functions of epithelial cells in different organs. Polarity in epithelial cells is characterized by different lipid and protein...
Cell Research 17, 100 - 116 (01 Feb 2007), doi: 10.1038/sj.cr.7310145, Original Article
23. Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers
G P Basten, S J Duthie, L Pirie,
SUMMARY: We have previously reported that supplementation with folic acid (1.2 mg day-1 for 12 week) elicited a significant improvement in the folate status of 61 healthy
CONTEXT: Epidemiological studies have consistently revealed an association between the consumption of fruit and vegetables and reduced incidence of cancers at various sites. Although the active dietary components have not been identified, folate...
British Journal of Cancer 94, 1942 - 1947 (19 Jun 2006), doi: 10.1038/sj.bjc.6603197, Genetics And Genomics
24. Brostallicin (PNU-166196) – a new DNA minor groove binder that retains sensitivity in DNA mismatch repair-deficient tumour cells
A Fedier, C Fowst, J Tursi, C Geroni, U Haller,
SUMMARY: Defects in DNA mismatch repair (MMR) are associated with a predisposition to tumorigenesis and with drug resistance owing to high mutation rates and failure
CONTEXT: Minor groove binders (MGBs) represent an interesting class of anticancer agents, which have been shown to be highly effective in in vitro and in vivo preclinical tumour models unresponsive to other antineoplastic agents (Martin et al,...
British Journal of Cancer 89, 1559 - 1565 (20 Oct 2003), doi: 10.1038/sj.bjc.6601316, Experimental Therapeutics
May 8, 2008
DNA in Science
1.DNA from Pre-Clovis Human Coprolites in Oregon, North America
M. Thomas P. Gilbert,1* Dennis L. Jenkins,2* Anders Götherstrom,3 Nuria Naveran,4 Juan J. Sanchez,5 Michael Hofreiter,6 Philip Francis Thomsen,1 Jonas Binladen,1 Thomas F. G. Higham,7 Robert M. Yohe, II,8 Robert Parr,8 Linda Scott Cummings,9 Eske Willerslev1
The timing of the first human migration into the Americas and its relation to the appearance of the Clovis technological complex in North America at about 11,000 to 10,800 radiocarbon years before the present (14C years B.P.) remains contentious. We establish that humans were present at Paisley 5 Mile Point Caves, in south-central Oregon, by 12,300 14C years B.P., through the recovery of human mitochondrial DNA (mtDNA) from coprolites, directly dated by accelerator mass spectrometry. The mtDNA corresponds to Native American founding haplogroups A2 and B2. The dates of the coprolites are >1000 14C years earlier than currently accepted dates for the Clovis complex.
1 Centre for Ancient Genetics, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark.2 Museum of Natural and Cultural History, 1224 University of Oregon, Eugene, OR 97403-1224, USA.3 Department of Evolutionary Biology, Uppsala University, Norbyvagten 18D, 74236 Uppsala, Sweden.4 Instituto de Medicina Legal, Facultad de Medicina, University of Santiago de Compostela, San Francisco s/n 15782, Santiago de Compostela, Spain.5 National Institute of Toxicology and Forensic Science, Canary Islands Delegation, 38320 Tenerife, Spain.6 Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany.7 Research Laboratory for Archaeology and the History of Art, Dyson Perrins Building, South Parks Road, Oxford, OX1 3QY, UK.8 Department of Sociology/Anthropology, California State University, 9001 Stockdale Highway, Bakersfield, CA 93311, USA.9 Palaeo Research Institute, 2675 Youngfield Street, Golden, CO 80401, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: ewillerslev@bi.ku.dk
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2.ROS-Generating Mitochondrial DNA Mutations Can Regulate Tumor Cell Metastasis
Kaori Ishikawa,1,2,3* Keizo Takenaga,4,5* Miho Akimoto,5 Nobuko Koshikawa,4 Aya Yamaguchi,1 Hirotake Imanishi,1 Kazuto Nakada,1,2 Yoshio Honma,5 Jun-Ichi Hayashi1
Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.
1 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.2 Tsukuba Advanced Research Alliance Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.3 Japan Society for the Promotion of Science (JSPS), 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan.4 Division of Chemotherapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan.5 Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: jih45@sakura.cc.tsukuba.ac.jp
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3.Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions
Jean-Pierre Vartanian, Denise Guétard, Michel Henry, Simon Wain-Hobson*
Cytidine deaminases of the APOBEC3 family all have specificity for single-stranded DNA, which may become exposed during replication or transcription of double-stranded DNA. Three human APOBEC3A (hA3A), hA3B, and hA3H genes are expressed in keratinocytes and skin, leading us to determine whether genetic editing of human papillomavirus (HPV) DNA occurred. In a study of HPV1a plantar warts and HPV16 precancerous cervical biopsies, hyperedited HPV1a and HPV16 genomes were found. Strictly analogous results were obtained from transfection experiments with HPV plasmid DNA and the three nuclear localized enzymes: hA3A, hA3C, and hA3H. Thus, stochastic or transient overexpression of APOBEC3 genes may expose the genome to a broad spectrum of mutations that could influence the development of tumors.
Molecular Retrovirology Unit, Institut Pasteur, 28 Rue de Docteur Roux, 75724 Paris cedex 15, France.
* To whom correspondence should be addressed. E-mail: simon@pasteur.fr
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4.DNA From Fossil Feces Breaks Clovis Barrier
Michael Balter
An international team reports online in Science this week what some experts consider the strongest evidence yet for an earlier peopling of the Americas: 14,000-year-old ancient DNA from fossilized human excrement (coprolites), found in caves in south-central Oregon.
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5.Single-Molecule DNA Sequencing of a Viral Genome
Timothy D. Harris,1* Phillip R. Buzby,1 Hazen Babcock,1 Eric Beer,1 Jayson Bowers,1 Ido Braslavsky,2 Marie Causey,1 Jennifer Colonell,1 James DiMeo,1 J. William Efcavitch,1 Eldar Giladi,1 Jaime Gill,1 John Healy,1 Mirna Jarosz,1 Dan Lapen,1 Keith Moulton,1 Stephen R. Quake,3 Kathleen Steinmann,1 Edward Thayer,1 Anastasia Tyurina,1 Rebecca Ward,1 Howard Weiss,1 Zheng Xie1
The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.
1 Helicos BioSciences Corporation, One Kendall Square, Cambridge, MA 02139, USA.2 Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA.3 Department of Bioengineering, Stanford University, and Howard Hughes Medical Institute, Stanford, CA 94305, USA.
* To whom correspondence should be addressed. E-mail: tharris@helicosbio.com
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6.Nutritional Control of Reproductive Status in Honeybees via DNA Methylation
R. Kucharski,* J. Maleszka,* S. Foret, R. Maleszka
Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. We show that silencing the expression of DNA methyltransferase Dnmt3, a key driver of epigenetic global reprogramming, in newly hatched larvae led to a royal jelly–like effect on the larval developmental trajectory; the majority of Dnmt3 small interfering RNA–treated individuals emerged as queens with fully developed ovaries. Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.
Molecular Genetics and Evolution, ARC Centre for the Molecular Genetics of Development, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: maleszka@rsbs.anu.edu.au
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7.Proposal to 'Wikify' GenBank Meets Stiff Resistance
Elizabeth Pennisi
In a letter in this week's issue of Science, a group of mycologists urges GenBank to allow researchers who discover inaccuracies in the database to append corrections. GenBank, however, says such a fix would cause more problems than it solves.
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8.An Oncogene-Induced DNA Damage Model for Cancer Development
Thanos D. Halazonetis,1* Vassilis G. Gorgoulis,2 Jiri Bartek3
Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.
1 Department of Molecular Biology and Department of Biochemistry, University of Geneva, CH-1205 Geneva, Switzerland.2 Department of Histology and Embryology, School of Medicine, University of Athens, GR-11527 Athens, Greece.3 Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
* To whom correspondence should be addressed. E-mail: Thanos.Halazonetis@molbio.unige.ch
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9.DNA Assembles Materials From the Ground Up
Robert F. Service
On page 594 of this week's issue of Science, researchers report using DNA as tweezers to pick up compounds and place them where they're wanted. The technique could help researchers put chains of molecules together to answer questions such as how different enzymes work together in a series.
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10.DNA SEQUENCING:A Plan to Capture Human Diversity in 1000 Genomes
Jocelyn Kaiser
Over the next 3 years, an international team plans to create a massive new catalog containing the complete genome sequences of 1000 individuals. It will help fill out the list of new genetic markers for common diseases that came out in 2007.
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11.Control of Genic DNA Methylation by a jmjC Domain-Containing Protein in Arabidopsis thaliana
Hidetoshi Saze,* Akiko Shiraishi, Asuka Miura, Tetsuji Kakutani
Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.
Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
* To whom correspondence should be addressed. E-mail: hsaze@lab.nig.ac.jp
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12.DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression
Bruno Perillo,1* Maria Neve Ombra,1* Alessandra Bertoni,2 Concetta Cuozzo,3 Silvana Sacchetti,3 Annarita Sasso,2 Lorenzo Chiariotti,2 Antonio Malorni,1 Ciro Abbondanza,4 Enrico V. Avvedimento2
Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.
1 Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche (C.N.R.), 83100 Avellino, Italy.2 Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Università degli Studi "Federico II," 80131 Naples, Italy.3 Naples Oncogenomic Center, Centro di Ingegneria Genetica (CEINGE), Biotecnologie Avanzate, 80131 Naples, Italy.4 Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, 80138 Naples, Italy.
* These authors contributed equally to this paper.
To whom correspondence should be addressed. E-mail: perillo@unina.it (B.P.); avvedim@unina.it (E.V.A.)
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13.Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase
Nadine K. Kolas,1* J. Ross Chapman,2* Shinichiro Nakada,1* Jarkko Ylanko,1,3 Richard Chahwan,2 Frédéric D. Sweeney,1,3 Stephanie Panier,1 Megan Mendez,1 Jan Wildenhain,1 Timothy M. Thomson,4 Laurence Pelletier,1,3 Stephen P. Jackson,2 Daniel Durocher1,3
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.
1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G1X5, Ontario, Canada.2 The Wellcome Trust and Cancer Research UK Gurdon Institute, and the Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.4 Department of Molecular and Cellular Biology, Instituto de Biología Molecular de Barcelona calle Jordi Girona 18-26, 08034 Barcelona, Spain.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: durocher@mshri.on.ca (D.D.); s.jackson@gurdon.cam.ac.uk (S.P.J.)
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14.End-to-End Stacking and Liquid Crystal Condensation of 6– to 20–Base Pair DNA Duplexes
Michi Nakata,1* Giuliano Zanchetta,2* Brandon D. Chapman,3 Christopher D. Jones,1 Julie O. Cross,4 Ronald Pindak,3 Tommaso Bellini,2 Noel A. Clark1
Short complementary B-form DNA oligomers, 6 to 20 base pairs in length, are found to exhibit nematic and columnar liquid crystal phases, even though such duplexes lack the shape anisotropy required for liquid crystal ordering. Structural study shows that these phases are produced by the end-to-end adhesion and consequent stacking of the duplex oligomers into polydisperse anisotropic rod-shaped aggregates, which can order into liquid crystals. Upon cooling mixed solutions of short DNA oligomers, in which only a small fraction of the DNA present is complementary, the duplex-forming oligomers phase-separate into liquid crystal droplets, leaving the unpaired single strands in isotropic solution. In a chemical environment where oligomer ligation is possible, such ordering and condensation would provide an autocatalytic link whereby complementarity promotes the extended polymerization of complementary oligomers.
1 Department of Physics and Liquid Crystal Materials Research Center, University of Colorado, Boulder, CO 80309–0390, USA.2 Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Milano, Italy.3 National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY 11973, USA.4 Advanced Photon Source, Argonne National Laboratory, Argonne, IL60439, USA.
* These authors contributed equally to this work.
Deceased.
To whom correspondence should be addressed. E-mail: tommaso.bellini@unimi.it (T.B.); noel.clark@colorado.edu (N.A.C.)
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15.DNA Circuits Get Up to Speed
Roy Bar-Ziv
An amplification mechanism brings DNA circuits closer to practical applications.
The author is in the Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: roy.bar-ziv@weizmann.ac.il
Full Text » PDF »
16.A key aspect of electronic circuits is amplification or gain, so that low signals can be distinguished from any persistent background.
Zhang et al. (p. 1121; see the Perspective by Bar-Ziv) show how gain can be achieved in biochemical circuits. They have designed complex catalytic networks based on DNA in which the output oligonucleotides that are released go on to act as catalysts for other reactions. The process is designed to be entropy driven so that the pathways for reactions are well controlled and can be modified at will. Possible applications lie in the field of catalysis, sensor development, the development of enzyme-free alternative for the polymerase chain reaction, and the construction of nanomachines.
PDF »
17.Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA
David Yu Zhang,1 Andrew J. Turberfield,2 Bernard Yurke,3* Erik Winfree1
Artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components. We introduce a design strategy that allows a specified input oligonucleotide to catalyze the release of a specified output oligonucleotide, which in turn can serve as a catalyst for other reactions. This reaction, which is driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. We have constructed and characterized several circuits that amplify nucleic acid signals, including a feedforward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics.
1 Computation and Neural Systems, California Institute of Technology, MC 136-93, 1200 East California Boulevard, Pasadena, CA91125, USA.2 Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, UK.3 Bell Laboratories, Alcatel-Lucent, Murray Hill, NJ 07974, USA.
* Present address: Materials Science and Engineering Department, Boise State University, Boise, ID 83725, USA.
To whom correspondence should be addressed. E-mail: winfree@caltech.edu (E.W.); dzhang@dna.caltech.edu (D.Y.Z.)
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18.Bypass of DNA Lesions Generated During Anticancer Treatment with Cisplatin by DNA Polymerase
Aaron Alt,1* Katja Lammens,1,2* Claudia Chiocchini,1 Alfred Lammens,1,2 J. Carsten Pieck,1 David Kuch,1 Karl-Peter Hopfner,1,2 Thomas Carell1
DNA polymerase (Pol ) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis–syn thymine dimers. We present structural and biochemical analysis of how Pol copies Pt-GG–containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol to replicate across strongly distorting DNA lesions.
1 Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilians University, D-81377 Munich, Germany.2 Gene Center at the Department of Chemistry and Biochemistry, Ludwig Maximilians University, D-81377 Munich, Germany.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: hopfner@lmb.uni-muenchen.de (K.-P.H.); thomas.carell@cup.uni-muenchen.de (T.C.)
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19.Ancient DNA Reveals Neandertals With Red Hair, Fair Complexions
Elizabeth Culotta
A pigmentation gene from the bones of two Neandertals, reported online this week in Science (www.sciencemag.org/cgi/content/abstract/1147417), indicates that at least some Neandertals had pale skin and red hair, similar to some of the Homo sapiens who today inhabit their European homeland.
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20.Mitochondrial DNA as a Genomic Jigsaw Puzzle
William Marande and Gertraud Burger*
In mitochondria of the unicellular eukaryote Diplonema, genes are systematically fragmented into small pieces that are encoded on separate chromosomes, transcribed individually, and then concatenated into contiguous messenger RNA molecules
Department of Biochemistry, Université de Montréal, Montréal, Quebec H3T 1J4, Canada.
* To whom correspondence should be addressed. E-mail: Gertraud.Burger@UMontreal.ca
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21.Structure of a NHEJ Polymerase-Mediated DNA Synaptic Complex
Nigel C. Brissett,1* Robert S. Pitcher,1* Raquel Juarez,2 Angel J. Picher,2 Andrew J. Green,1 Timothy R. Dafforn,3 Gavin C. Fox,4 Luis Blanco,2 Aidan J. Doherty1
Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway required to maintain genome stability. Many prokaryotes possess a minimalist NHEJ apparatus required to repair DSBs during stationary phase, composed of two conserved core proteins, Ku and ligase D (LigD). The crystal structure of Mycobacterium tuberculosis polymerase domain of LigD mediating the synapsis of two noncomplementary DNA ends revealed a variety of interactions, including microhomology base pairing, mismatched and flipped-out bases, and 3' termini forming hairpin-like ends. Biochemical and biophysical studies confirmed that polymerase-induced end synapsis also occurs in solution. We propose that this DNA synaptic structure reflects an intermediate bridging stage of the NHEJ process, before end processing and ligation, with both the polymerase and the DNA sequence playing pivotal roles in determining the sequential order of synapsis and remodeling before end joining.
1 Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK.2 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, 28049 Madrid, Spain.3 Department of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.4 European Synchrotron Radiation Facility, LLS-BM16, Grenoble Cedex 9, France.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: lblanco@cbm.uam.es (L.B.); ajd21@sussex.ac.uk (A.J.D.)
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22.UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells
Magnolia Bostick,1* Jong Kyong Kim,2* Pierre-Olivier Estève,2 Amander Clark,1 Sriharsa Pradhan,2 Steven E. Jacobsen1,3
Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.
1 Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.2 New England BioLabs, Ipswich, MA 01938, USA.3 Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: pradhan@neb.com (S.P.); jacobsen@ucla.edu (S.E.J.)
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M. Thomas P. Gilbert,1* Dennis L. Jenkins,2* Anders Götherstrom,3 Nuria Naveran,4 Juan J. Sanchez,5 Michael Hofreiter,6 Philip Francis Thomsen,1 Jonas Binladen,1 Thomas F. G. Higham,7 Robert M. Yohe, II,8 Robert Parr,8 Linda Scott Cummings,9 Eske Willerslev1
The timing of the first human migration into the Americas and its relation to the appearance of the Clovis technological complex in North America at about 11,000 to 10,800 radiocarbon years before the present (14C years B.P.) remains contentious. We establish that humans were present at Paisley 5 Mile Point Caves, in south-central Oregon, by 12,300 14C years B.P., through the recovery of human mitochondrial DNA (mtDNA) from coprolites, directly dated by accelerator mass spectrometry. The mtDNA corresponds to Native American founding haplogroups A2 and B2. The dates of the coprolites are >1000 14C years earlier than currently accepted dates for the Clovis complex.
1 Centre for Ancient Genetics, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark.2 Museum of Natural and Cultural History, 1224 University of Oregon, Eugene, OR 97403-1224, USA.3 Department of Evolutionary Biology, Uppsala University, Norbyvagten 18D, 74236 Uppsala, Sweden.4 Instituto de Medicina Legal, Facultad de Medicina, University of Santiago de Compostela, San Francisco s/n 15782, Santiago de Compostela, Spain.5 National Institute of Toxicology and Forensic Science, Canary Islands Delegation, 38320 Tenerife, Spain.6 Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany.7 Research Laboratory for Archaeology and the History of Art, Dyson Perrins Building, South Parks Road, Oxford, OX1 3QY, UK.8 Department of Sociology/Anthropology, California State University, 9001 Stockdale Highway, Bakersfield, CA 93311, USA.9 Palaeo Research Institute, 2675 Youngfield Street, Golden, CO 80401, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: ewillerslev@bi.ku.dk
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2.ROS-Generating Mitochondrial DNA Mutations Can Regulate Tumor Cell Metastasis
Kaori Ishikawa,1,2,3* Keizo Takenaga,4,5* Miho Akimoto,5 Nobuko Koshikawa,4 Aya Yamaguchi,1 Hirotake Imanishi,1 Kazuto Nakada,1,2 Yoshio Honma,5 Jun-Ichi Hayashi1
Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.
1 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.2 Tsukuba Advanced Research Alliance Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.3 Japan Society for the Promotion of Science (JSPS), 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan.4 Division of Chemotherapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan.5 Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: jih45@sakura.cc.tsukuba.ac.jp
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3.Evidence for Editing of Human Papillomavirus DNA by APOBEC3 in Benign and Precancerous Lesions
Jean-Pierre Vartanian, Denise Guétard, Michel Henry, Simon Wain-Hobson*
Cytidine deaminases of the APOBEC3 family all have specificity for single-stranded DNA, which may become exposed during replication or transcription of double-stranded DNA. Three human APOBEC3A (hA3A), hA3B, and hA3H genes are expressed in keratinocytes and skin, leading us to determine whether genetic editing of human papillomavirus (HPV) DNA occurred. In a study of HPV1a plantar warts and HPV16 precancerous cervical biopsies, hyperedited HPV1a and HPV16 genomes were found. Strictly analogous results were obtained from transfection experiments with HPV plasmid DNA and the three nuclear localized enzymes: hA3A, hA3C, and hA3H. Thus, stochastic or transient overexpression of APOBEC3 genes may expose the genome to a broad spectrum of mutations that could influence the development of tumors.
Molecular Retrovirology Unit, Institut Pasteur, 28 Rue de Docteur Roux, 75724 Paris cedex 15, France.
* To whom correspondence should be addressed. E-mail: simon@pasteur.fr
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4.DNA From Fossil Feces Breaks Clovis Barrier
Michael Balter
An international team reports online in Science this week what some experts consider the strongest evidence yet for an earlier peopling of the Americas: 14,000-year-old ancient DNA from fossilized human excrement (coprolites), found in caves in south-central Oregon.
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5.Single-Molecule DNA Sequencing of a Viral Genome
Timothy D. Harris,1* Phillip R. Buzby,1 Hazen Babcock,1 Eric Beer,1 Jayson Bowers,1 Ido Braslavsky,2 Marie Causey,1 Jennifer Colonell,1 James DiMeo,1 J. William Efcavitch,1 Eldar Giladi,1 Jaime Gill,1 John Healy,1 Mirna Jarosz,1 Dan Lapen,1 Keith Moulton,1 Stephen R. Quake,3 Kathleen Steinmann,1 Edward Thayer,1 Anastasia Tyurina,1 Rebecca Ward,1 Howard Weiss,1 Zheng Xie1
The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.
1 Helicos BioSciences Corporation, One Kendall Square, Cambridge, MA 02139, USA.2 Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA.3 Department of Bioengineering, Stanford University, and Howard Hughes Medical Institute, Stanford, CA 94305, USA.
* To whom correspondence should be addressed. E-mail: tharris@helicosbio.com
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6.Nutritional Control of Reproductive Status in Honeybees via DNA Methylation
R. Kucharski,* J. Maleszka,* S. Foret, R. Maleszka
Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. We show that silencing the expression of DNA methyltransferase Dnmt3, a key driver of epigenetic global reprogramming, in newly hatched larvae led to a royal jelly–like effect on the larval developmental trajectory; the majority of Dnmt3 small interfering RNA–treated individuals emerged as queens with fully developed ovaries. Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.
Molecular Genetics and Evolution, ARC Centre for the Molecular Genetics of Development, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: maleszka@rsbs.anu.edu.au
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7.Proposal to 'Wikify' GenBank Meets Stiff Resistance
Elizabeth Pennisi
In a letter in this week's issue of Science, a group of mycologists urges GenBank to allow researchers who discover inaccuracies in the database to append corrections. GenBank, however, says such a fix would cause more problems than it solves.
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8.An Oncogene-Induced DNA Damage Model for Cancer Development
Thanos D. Halazonetis,1* Vassilis G. Gorgoulis,2 Jiri Bartek3
Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.
1 Department of Molecular Biology and Department of Biochemistry, University of Geneva, CH-1205 Geneva, Switzerland.2 Department of Histology and Embryology, School of Medicine, University of Athens, GR-11527 Athens, Greece.3 Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
* To whom correspondence should be addressed. E-mail: Thanos.Halazonetis@molbio.unige.ch
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9.DNA Assembles Materials From the Ground Up
Robert F. Service
On page 594 of this week's issue of Science, researchers report using DNA as tweezers to pick up compounds and place them where they're wanted. The technique could help researchers put chains of molecules together to answer questions such as how different enzymes work together in a series.
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10.DNA SEQUENCING:A Plan to Capture Human Diversity in 1000 Genomes
Jocelyn Kaiser
Over the next 3 years, an international team plans to create a massive new catalog containing the complete genome sequences of 1000 individuals. It will help fill out the list of new genetic markers for common diseases that came out in 2007.
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11.Control of Genic DNA Methylation by a jmjC Domain-Containing Protein in Arabidopsis thaliana
Hidetoshi Saze,* Akiko Shiraishi, Asuka Miura, Tetsuji Kakutani
Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.
Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
* To whom correspondence should be addressed. E-mail: hsaze@lab.nig.ac.jp
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12.DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression
Bruno Perillo,1* Maria Neve Ombra,1* Alessandra Bertoni,2 Concetta Cuozzo,3 Silvana Sacchetti,3 Annarita Sasso,2 Lorenzo Chiariotti,2 Antonio Malorni,1 Ciro Abbondanza,4 Enrico V. Avvedimento2
Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.
1 Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche (C.N.R.), 83100 Avellino, Italy.2 Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Università degli Studi "Federico II," 80131 Naples, Italy.3 Naples Oncogenomic Center, Centro di Ingegneria Genetica (CEINGE), Biotecnologie Avanzate, 80131 Naples, Italy.4 Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, 80138 Naples, Italy.
* These authors contributed equally to this paper.
To whom correspondence should be addressed. E-mail: perillo@unina.it (B.P.); avvedim@unina.it (E.V.A.)
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13.Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase
Nadine K. Kolas,1* J. Ross Chapman,2* Shinichiro Nakada,1* Jarkko Ylanko,1,3 Richard Chahwan,2 Frédéric D. Sweeney,1,3 Stephanie Panier,1 Megan Mendez,1 Jan Wildenhain,1 Timothy M. Thomson,4 Laurence Pelletier,1,3 Stephen P. Jackson,2 Daniel Durocher1,3
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.
1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G1X5, Ontario, Canada.2 The Wellcome Trust and Cancer Research UK Gurdon Institute, and the Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.4 Department of Molecular and Cellular Biology, Instituto de Biología Molecular de Barcelona calle Jordi Girona 18-26, 08034 Barcelona, Spain.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: durocher@mshri.on.ca (D.D.); s.jackson@gurdon.cam.ac.uk (S.P.J.)
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14.End-to-End Stacking and Liquid Crystal Condensation of 6– to 20–Base Pair DNA Duplexes
Michi Nakata,1* Giuliano Zanchetta,2* Brandon D. Chapman,3 Christopher D. Jones,1 Julie O. Cross,4 Ronald Pindak,3 Tommaso Bellini,2 Noel A. Clark1
Short complementary B-form DNA oligomers, 6 to 20 base pairs in length, are found to exhibit nematic and columnar liquid crystal phases, even though such duplexes lack the shape anisotropy required for liquid crystal ordering. Structural study shows that these phases are produced by the end-to-end adhesion and consequent stacking of the duplex oligomers into polydisperse anisotropic rod-shaped aggregates, which can order into liquid crystals. Upon cooling mixed solutions of short DNA oligomers, in which only a small fraction of the DNA present is complementary, the duplex-forming oligomers phase-separate into liquid crystal droplets, leaving the unpaired single strands in isotropic solution. In a chemical environment where oligomer ligation is possible, such ordering and condensation would provide an autocatalytic link whereby complementarity promotes the extended polymerization of complementary oligomers.
1 Department of Physics and Liquid Crystal Materials Research Center, University of Colorado, Boulder, CO 80309–0390, USA.2 Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Milano, Italy.3 National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY 11973, USA.4 Advanced Photon Source, Argonne National Laboratory, Argonne, IL60439, USA.
* These authors contributed equally to this work.
Deceased.
To whom correspondence should be addressed. E-mail: tommaso.bellini@unimi.it (T.B.); noel.clark@colorado.edu (N.A.C.)
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15.DNA Circuits Get Up to Speed
Roy Bar-Ziv
An amplification mechanism brings DNA circuits closer to practical applications.
The author is in the Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: roy.bar-ziv@weizmann.ac.il
Full Text » PDF »
16.A key aspect of electronic circuits is amplification or gain, so that low signals can be distinguished from any persistent background.
Zhang et al. (p. 1121; see the Perspective by Bar-Ziv) show how gain can be achieved in biochemical circuits. They have designed complex catalytic networks based on DNA in which the output oligonucleotides that are released go on to act as catalysts for other reactions. The process is designed to be entropy driven so that the pathways for reactions are well controlled and can be modified at will. Possible applications lie in the field of catalysis, sensor development, the development of enzyme-free alternative for the polymerase chain reaction, and the construction of nanomachines.
PDF »
17.Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA
David Yu Zhang,1 Andrew J. Turberfield,2 Bernard Yurke,3* Erik Winfree1
Artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components. We introduce a design strategy that allows a specified input oligonucleotide to catalyze the release of a specified output oligonucleotide, which in turn can serve as a catalyst for other reactions. This reaction, which is driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. We have constructed and characterized several circuits that amplify nucleic acid signals, including a feedforward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics.
1 Computation and Neural Systems, California Institute of Technology, MC 136-93, 1200 East California Boulevard, Pasadena, CA91125, USA.2 Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, UK.3 Bell Laboratories, Alcatel-Lucent, Murray Hill, NJ 07974, USA.
* Present address: Materials Science and Engineering Department, Boise State University, Boise, ID 83725, USA.
To whom correspondence should be addressed. E-mail: winfree@caltech.edu (E.W.); dzhang@dna.caltech.edu (D.Y.Z.)
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18.Bypass of DNA Lesions Generated During Anticancer Treatment with Cisplatin by DNA Polymerase
Aaron Alt,1* Katja Lammens,1,2* Claudia Chiocchini,1 Alfred Lammens,1,2 J. Carsten Pieck,1 David Kuch,1 Karl-Peter Hopfner,1,2 Thomas Carell1
DNA polymerase (Pol ) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis–syn thymine dimers. We present structural and biochemical analysis of how Pol copies Pt-GG–containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol to replicate across strongly distorting DNA lesions.
1 Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilians University, D-81377 Munich, Germany.2 Gene Center at the Department of Chemistry and Biochemistry, Ludwig Maximilians University, D-81377 Munich, Germany.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: hopfner@lmb.uni-muenchen.de (K.-P.H.); thomas.carell@cup.uni-muenchen.de (T.C.)
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19.Ancient DNA Reveals Neandertals With Red Hair, Fair Complexions
Elizabeth Culotta
A pigmentation gene from the bones of two Neandertals, reported online this week in Science (www.sciencemag.org/cgi/content/abstract/1147417), indicates that at least some Neandertals had pale skin and red hair, similar to some of the Homo sapiens who today inhabit their European homeland.
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20.Mitochondrial DNA as a Genomic Jigsaw Puzzle
William Marande and Gertraud Burger*
In mitochondria of the unicellular eukaryote Diplonema, genes are systematically fragmented into small pieces that are encoded on separate chromosomes, transcribed individually, and then concatenated into contiguous messenger RNA molecules
Department of Biochemistry, Université de Montréal, Montréal, Quebec H3T 1J4, Canada.
* To whom correspondence should be addressed. E-mail: Gertraud.Burger@UMontreal.ca
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21.Structure of a NHEJ Polymerase-Mediated DNA Synaptic Complex
Nigel C. Brissett,1* Robert S. Pitcher,1* Raquel Juarez,2 Angel J. Picher,2 Andrew J. Green,1 Timothy R. Dafforn,3 Gavin C. Fox,4 Luis Blanco,2 Aidan J. Doherty1
Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway required to maintain genome stability. Many prokaryotes possess a minimalist NHEJ apparatus required to repair DSBs during stationary phase, composed of two conserved core proteins, Ku and ligase D (LigD). The crystal structure of Mycobacterium tuberculosis polymerase domain of LigD mediating the synapsis of two noncomplementary DNA ends revealed a variety of interactions, including microhomology base pairing, mismatched and flipped-out bases, and 3' termini forming hairpin-like ends. Biochemical and biophysical studies confirmed that polymerase-induced end synapsis also occurs in solution. We propose that this DNA synaptic structure reflects an intermediate bridging stage of the NHEJ process, before end processing and ligation, with both the polymerase and the DNA sequence playing pivotal roles in determining the sequential order of synapsis and remodeling before end joining.
1 Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK.2 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, 28049 Madrid, Spain.3 Department of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.4 European Synchrotron Radiation Facility, LLS-BM16, Grenoble Cedex 9, France.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: lblanco@cbm.uam.es (L.B.); ajd21@sussex.ac.uk (A.J.D.)
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22.UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells
Magnolia Bostick,1* Jong Kyong Kim,2* Pierre-Olivier Estève,2 Amander Clark,1 Sriharsa Pradhan,2 Steven E. Jacobsen1,3
Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.
1 Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.2 New England BioLabs, Ipswich, MA 01938, USA.3 Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: pradhan@neb.com (S.P.); jacobsen@ucla.edu (S.E.J.)
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