DNA (Gene) Transformation Protocols

Electrotransformation (Crawford Lab.)

PCR screening of transformants (Crawford Lab.)

Bacterial Transformation and Transfection (Roe Lab.)

Preparation of Competent Cells

Competent cell preparation

Method: Transformation of Plasmids/Cosmids into E. coli (Helen Donis-Keller Lab.)

Ligations in Low Melting Temperature Agarose (Helen Donis-Keller Lab)

Transformation of E. coli by electroporation (UMBC)

Electrocompetent E. coli preparation (Hahn Lab.)

Rubidium Chloride Method, NEB (BioLabs)

CaCl2 TRANSFORMATION OF E. coli (Hancock Lab.)

DNA Electroporation (Stanford DNA Center)

Bacterial Transformation (University of Arizona)

Transformation of E. coli with pGAL™ (blue colony)

Transformation of Bacteria by Plasmid DNA (Laboratoryexperimets.com)

Transformation of E. coli by Electroporation (Stanford)

Protocol - Chemical Transformation protocol (Baker Lab)

Transformation of Competent Cells (Laurie Lab)

Transformation Protocol for Arabidopsis - OpenWetWare

PCR Mutagenesis/Co-Transformation Protocol

Chen Lab Protocol--transformation

Chen Lab Protocol--transformation

High Efficiency Transformationmethod Page (TRAFO Page)

Yeast Transformation Protocol (by Sudhir Nayak)

Transformation Protocol (Laurie Lab.)

High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures

http://www.biocompare.com/technicalarticle/1137/High-Electrotransformation-Efficiencies-Obtained-With-DNA-From-Ligation-Mixtures-from-Bio-Rad.html

Selected Articles on Mutagenesis

MW Unger , SY Liu , and DE Rancourt

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination
Nucl. Acids Res. 27: 1480-1484.

Paul Gaytán , Jorge Yáñez , Filiberto Sánchez , and Xavier Soberón

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols
Nucl. Acids Res. 29: e9.

RD Kirsch , and E Joly

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes
Nucl. Acids Res. 26: 1848-1850.

Qing Lin , Sarah L. Donahue , Tracy Moore-Jarrett , Shang Cao , Anna B. Osipovich , and H. Earl Ruley

Mutagenesis of diploid mammalian genes by gene entrapment
Nucleic Acids Research Advance Access published on November 6, 2006, DOI 10.1093/nar/gkl728.
Nucl. Acids Res. 34: e139.

Igor Shevelev , Giuseppina Blanca , Giuseppe Villani , Kristijan Ramadan , Silvio Spadari , Ulrich Hübscher , and Giovanni Maga

Mutagenesis of human DNA polymerase : essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities
Nucl. Acids Res. 31: 6916-6925.

Jaesung Lee , and David L. Herrin

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth
Nucl. Acids Res. 31: 4361-4372.

Michael O’Connor , Wyan-Ching Mimi Lee , Anuj Mankad , Catherine L. Squires , and Albert E. Dahlberg

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable
Nucl. Acids Res. 29: 710-715.

Eun Ju Lee , Joel Glasgow , Sew-Fen Leu , Ali O. Belduz , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 the cyclic nucleotide binding pocket
Nucl. Acids Res. 22: 2894-2901.

M.J. Pillaire , A. Margot , G. Villani , A. Sarasin , M. Defias , and A. Gentil

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(ll) adduct placed on codon 13 of the human H-ras proto-oncogen
Nucl. Acids Res. 22: 2519-2524.

Pamela Anderson , Joseph Monforte , Richard Tritz , Steven Nesbitt , John Hearst , and Arnold Hamper

Mutagenesis of the hairpin ribozyme
Nucl. Acids Res. 22: 1096-1100.

Huey-Nan Wu , Jun-YU Lee , Huey-Wen Huang , Yi-shuian Huang , and Tung-Guang Hsueh

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme
Nucl. Acids Res. 21: 4193-4199.

Ali O. Belduz , Eun Ju Lee , and James G. Harman

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli targeting positions 72 and 82 of the cyclic nucleotide binding pocket
Nucl. Acids Res. 21: 1827-1835.

Huey-Nan Wu , and Zhi-Shun Huang

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA
Nucl. Acids Res. 20: 5937-5941.

V. Pletsa , A. Gentil , A. Margot , J. Armier , S.A. Kyrtopoulos , and A. Sarasin

Mutagenesis by 06 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells
Nucl. Acids Res. 20: 4897-4901.

Akira Sekiguchi , Yasuo Komatsu , Makoto Koizumi , and Eiko Ohtsuka

Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco rinspot virus and its binding to polyamines
Nucl. Acids Res. 19: 6833-6838.

Aidan J. Doherty , Andrew F. Worall , and Bernard A. Connolly

Mutagenesis of the DNA binding residues in bovine pancreatic DNase1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease
Nucl. Acids Res. 19: 6129-6132.

Christian Baron , Johann Heider , and August Böck

Mutagenesis of selC, the gene for the selenocysteine-insertlng tRNA-species in E.coli: effects on in vivo function
Nucl. Acids Res. 18: 6761-6766.

Candice C. Sheldon , and Robert H. Symons

Mutagenesis analysis of a self-cleaving RNA
Nucl. Acids Res. 17: 5679-5685.

Jacques Piette , Howard B. Gamper , Albert Van de Vorst , and John E. Hearst

Mutagenesis induced by site specifically placed 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts
Nucl. Acids Res. 16: 9961-9977.

G. Chua , L. Taricani , W. Stangle , and P. G. Young

Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe
Nucl. Acids Res. 28: e53.

MJ Singer , MA Podyminogin , MA Metcalf , MW Reed , DA Brown , HB Gamper , RB Meyer , and RM Wydro

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides
Nucl. Acids Res. 27: e38.

A Melnikov , and PJ Youngman

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
Nucl. Acids Res. 27: 1056-1062.

RM Tucker , and DT Burke

Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol
Nucl. Acids Res. 24: 3467-3468.

David P. Siderovski , Toshifumi Matsuyama , Elena Frigerio , Stephen Chui , Xia Min , Heather Erfle , Martin Sumner-Smith , Richard W. Bamett , and Tak W. Mak

Random mutagenesis of the human immunodeficiency virus type-1 frans-activator of transcription (HIV-1 Tat)
Nucl. Acids Res. 20: 5311-5320.

Xiubei Liao , David Selinger , Steven Althoff , Anne Chiang , Diana Hamilton , Min Ma , and Jo Ann Wise

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites
Nucl. Acids Res. 20: 1607-1615.

Yuhong Zhou , Xiaoping Zhang , and Richard H. Ebright

Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase
Nucl. Acids Res. 19: 6052.

Bruno A. Gaëta , Stephen J. Sharp , and Thomas S. Stewart

Saturation mutagenesis of the Drosophila tRNAArg gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation
Nucl. Acids Res. 18: 1541-1548.

Laurent Dulau , Alain Cheyrou , and Michel Aigle

Directed mutagenesis using PCR
Nucl. Acids Res. 17: 2873.

John M. Hinz , Robert S. Tebbs , Paul F. Wilson , Peter B. Nham , Edmund P. Salazar , Hatsumi Nagasawa , Salustra S. Urbin , Joel S. Bedford , and Larry H. Thompson

Repression of mutagenesis by Rad51D-mediated homologous recombination
Nucl. Acids Res. 34: 1358-1368.

Tuck Seng Wong , Kang Lan Tee , Berhard Hauer , and Ulrich Schwaneberg

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution
Nucl. Acids Res. 32: e26.

Gordana Maravic , Janusz M. Bujnicki , Marcin Feder , Sándor Pongor , and Mirna Flögel

Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein–RNA interactions
Nucl. Acids Res. 31: 4941-4949.

Fumi Nagatsugi , Shigeki Sasaki , Paul S. Miller , and Michael M. Seidman

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog
Nucl. Acids Res. 31: e31.

Assen Marintchev , Michael R. Gryk , and Gregory P. Mullen

Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase ß
Nucl. Acids Res. 31: 580-588.

P. Soultanas , and D. B. Wigley

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus
Nucl. Acids Res. 30: 4051-4060.

MS Dillingham , P Soultanas , and DB Wigley

Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation
Nucl. Acids Res. 27: 3310-3317.

P Neuner , R Cortese , and P Monaci

Codon-based mutagenesis using dimer-phosphoramidites
Nucl. Acids Res. 26: 1223-1227.

B Hallet , DJ Sherratt , and F Hayes

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein
Nucl. Acids Res. 25: 1866-1867.

W Wu , C Palaniappan , RA Bambara , and PJ Fay

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro
Nucl. Acids Res. 24: 1710-1718.

Gil Barzilay , Lisa J. Walker , Craig N. Robson , and Ian D. Hickson

Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity
Nucl. Acids Res. 23: 1544-1550.

Chung-Nam Chung , Yasushi Hamaguchi , Tasuku Honjo , and Masashi Kawaichi

Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jx
Nucl. Acids Res. 22: 2938-2944.

DNA Forensics -- Read Interesting Cases

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.)]

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.

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.

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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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11. Structure of the Elk-1–DNA complex reveals how DNA-distal residues affect ETS domain recognition of DNA

Yi Mo, Benjamin Vaessen, Karen Johnston, Ronen Marmorstein

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

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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

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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

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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

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15. Genetic interaction between DNA polymerase β and DNA-PKcs in embryogenesis and neurogenesis

N Niimi, N Sugo, Y Aratani, H Koyama

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

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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

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17. DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice

Petra Hanke, Matthias Serwe, Frank Dombrowski, Tilman Sauerbruch, Wolfgang H Caselmann

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

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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

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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

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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

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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

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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

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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, N Vaughan, M H Hill, H J Powers

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

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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, S Marchini, D Fink

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

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