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
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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.
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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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May 8, 2008
DNA in Science
May 6, 2008
Modern tools for identification of nucleic acid-binding proteins
Abstract
Numerous biological mechanisms depend on nucleic acid–protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.
Keywords: Nucleic acid-binding protein; Protein library; Affinity chromatography; Multiprotein complexes
DNA Two-Dimensional Polyacrylamide Gel Electrophoresis
2D-Gel electrophoresis (2DE)and proteomics: an online tutorial and practical ... solubilization for separation using two-dimensional gel electrophoresis. ...
www.aber.ac.uk/~mpgwww/Proteome/Tut_2D.html
Two-dimensional polyacrylamide gel electrophoresis database, including manual for mapping proteomes of different phyla.
expasy.org/ch2d/
Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein ...
www.proteomesci.com/content/2/1/6
Charge (IEF) and size (SDS-protein complex) separations can be combined in a two-dimensional approach. Two-dimensional gel electrophoresis is one of the ...
www.britannica.com/eb/topic-611230/two-dimensional-gel-electrophoresis
Two-dimensional gel electrophoresis as tool for. proteomics studies in combination with protein. identification by mass spectrometry ...
doi.wiley.com/10.1002/pmic.200500874
Springer Protocols is the largest subscription-based electronic database of reproducible ... Sample preparation for two-dimensional gel electrophoresis. ...
www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-463-6_8
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular ... This four-day protocol provides detailed information on how to apply ...
www.natureprotocols.com/2006/07/20/stateoftheart_twodimensional_g.php
Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA ... Here we provide a protocol for a simple approach that uses these ...
www.natureprotocols.com/2007/07/26/twodimensional_gel_electrophor.php
A Solubility Optimization Protocol for Two-Dimensional Gel Electrophoresis of Cardiac Tissue. By: Brian A. Stanley3 4, Jennifer E. Van Eyk3 4 5 6 ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59745-214-9:59
Two dimensional gel electrophoresis of proteins [View Printable] ... this by two dimentional gel electrophoresis method. Does anyone have a good protocol? ...
www.scientistsolutions.com/t585-Two+dimensional+gel+electrophoresis+of+proteins.html
Right arrow, Electrophoresis of Proteins. Right arrow, Related Protocols ... of Maize Embryo Proteins for Two-Dimensional Gel Electrophoresis Using a ...
www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4235
Two-dimensional gel electrophoresis is the combination of two ... A third alternate protocol describes how two-dimensional electrophoresis can be performed ...
https:/.../index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=9E662A36DC81D6EAF62978AC355691F1
This protocol was adapted from “Two-dimensional gel electrophoresis of proteins,” in Cells: A Laboratory Manual (eds. Spector, D.L., ...
npg.nature.com/nmeth/journal/v2/n1/pdf/nmeth0105-83.p
Hurkman, W.J., Tanaka, C.K. (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81:802-806. ...
proteomics.missouri.edu/aboutus/TechTips.doc
Fractionation Of Human Plasma Proteins For Two-Dimensional Gel Electrophoresis Using A Multicompartment Electrolyzer MCE -Subscription Required Protocol.
www.biocompare.com/protocols/protocol.asp?id=2145
protocols to resolve proteins based on conventional carrier ampholytes. ..... DNA sequence data: usefulness of two-dimensional gel electrophoresis and ...
proteomics.cancer.dk/images/protocols/Gel-based%20proteomics.pdf
The two-dimensional gel electrophoresis described here readily resolved linear, .... Methods in Molecular Biology: DNA Topoisomerase Protocols. ...
www.pubmedcentral.nih.gov/articlerender.fcgi?artid=55791
The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the ..... TROUBLESHOOTING. Troubleshooting advice can be found in Table 1. ...
www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2001252
Apr 23, 2008
Laboratory Manual for Biomedical Research
Laboratory Manual for Biomedical Research
New Edition
Chapter 1. General Lab Techniques
Lab security and basic techniques
Advanced lab skills
Advanced lab skills (2)
Chapter 2. Molecular Biology -- Molecular Separation
Molecular Separation
DNA Agarose Gel Electrophoresis
RNA Agarose Gel Electrophoresis
SDS-Page - Polyacrylamide gel electrophoresis
2D Page-Two - Dimensional Polyacrylamide Gel Electrophoresis
Ion Exchange Chromatography
Gel Filtration Chromatography
Affinity chromatography
Chapter 3. Molecular Biology -- DNA and RNA
Nucleic acid methods (1)
Nucleic acid methods (2)
DNA isolation & related protocols
DNA Purification (glass milk vs electroelution)
DNA, RNA Sequencing
RNA Isolation and Purification
Isolation of DNA,RNA, and Protein simultaneously.
DNA mutation detection by SSCP
Preparation of DNA and RNA probes
Southern blot hybridization
Northern blot hybridization
Loss of Heterozygosity (LOH)
Gene knockout protocol
SiRNA gene knockout
Plasmid and its usefulness
DNA library construction
Microarray protocols.
Basic knowledge of microarray.
Introduction to Microarray.
MicroArray Procedure
Total RNA Isolation from cultured cells.
DNase Treatment of Total RNA
Making the single strand cDNA probe.
Automated Slide Processor (ASP) Version for hybridization.
Washing microarrays in ASP.
Processing of Array slide
Pre-hybridization of the processed slides (NON-Automated version).
Hybridization of Cy3 + Cy5 probe to glass array (NON-Automated version).
Preparation of Dendrimer Cy3 and Cy5.
Washing unbound probe from glass array (NON-Automated version).
Hybridization of Dendrimers (Cy3 and Cy5) to Array (NON-Automated version).
Washing unbound dendrimer from glass array(NON-Automated version).
Microarray Dababases
Troubleshooting
Other Microarray Protocols (1,2)
Gene transfection
Gene therapy for cancer
Molecular cloning
Conditional gene transfection(Tet on/off)
Chapter 4. Genetics
Epigenetics protocols
Mutagenesis protocols
Single nucleotide polymorphisms (SNPs)
Chapter 5. Molecular Biology --PCR Serials
PCR,RT-PCR,Real time PCR etc.
PCR (General Procedure)
Recommended Reagent Concentrations
Recommended Reaction Conditions
Initial Conditions
Temperature Cycling
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labelling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
AND ALWAYS REMEMBER
PCR Primer Design Tools
RT-PCR
Real time PCR
More PCR Protocols Online
Video and Animation of PCR
Mouse Genotyping by PCR
PCR Based Molecular Cloning
PCR Troubleshootings
Chapter 6. Molecular Biology – Protein
Protein methods
Protein labeling techniques
Protein sequencing
Subcellular fractionations
Western blot hybridization
Western Blot Hybridization Center
Western Blot Full Procedure (from Pierce)
Western Blotting Procedure with Video Show
Protocol 1: Western Blot
Sample protein preparation
Protein concentration assay
Electrophoresis and blotting
Blocking non-specific antigen
Incubation with primary antibody
Incubation with secondary antibody
Substrate (ECL) incubation
Restore a western blot using pierce stripping buffer
Western blot related solutions and buffers
Western blot handbook
Western blot troubleshooting
Protocol 2: Western Blot 2.
Protocol 3: Western Blot 3.
More online Western blot protocols (1) (2)
Protein chips
Kinase assay
Methods for detecting protein phosphorylation
Chapter 7. DNA Protein Interactions
EMSA
ChIP assay
Chapter 8. Immunohistochemistry/immunology
Immunology/Histology
Preparing silanized (+plus) slides
Microscopy Techniques
Confocal microscope technique
Electron microscopy
HE staining
DAPI & PI nucleic acid stain
Hybridization in situ
FISH
Special cell & cell fraction stains
Antibody purification
Antibody storage and handling
Conjugation of monoclonal antibodies
Antigen retrieval
Immunoperoxidase staining techniques
Immunofluorohistochemistry
Immunoprecipitation
Histotechnology--technical methods
FRQs for histochemistry
Laser Capture Microdissection
Elisa
Chapter 9. Cellular Biology
General cell culture protocols
Chromosome karyotype
Proliferation assays (MTT, BrdU, 3H-Thymidine incoporation)
Cell cycle assay
Migration assay
Stem cell & related protocols
Introduction
What are the unique properties of all stem cells?
What are embryonic stem cells?
What are adult stem cells?
What are the similarities and differences between embryonic and adult stem cells?
What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized?
Where can I get more information?
International Society for Stem Cell Research (ISSCR)
Culture of Human Embryonic Stem Cells (hESC)
Establishment and characterization of human embryonic stem cell line
Rat stem cell isolation and culture
Mouse stem cell isolation and culture
Induction of Stem Cell Differentiation
Cancer stem cells
Video Data on Stem Cell Research (click to show)
Apoptosis and related protocols
Soft Agar Assay for Colony Formation
Aorta ring assay
GFP transfection
Blood cell fractionation (white blood cell isolation)
Endothelial cell isolation and culture protocols
Flow cytometry (FCM)
Chapter 10. Metabolism, GC/MS, NMR and Proteomics
GC/MS Background
Glucose metabolism and its related protocols
PAS staining
Deoxyribose procedure
Ribose metabolism analysis
Polysaccharide sequencing
Lactate cleanup and derivative
Amino acids
Fatty acids
Lipid protocols
Cholesterol
Bile acids
Urea procedure
Choline Incorporation Assay
Isotope Ratio Mass Spectrometer (IRMS)
Liquid chromatography / mass spectrometer (LC/MS)
Proteomics
NMR protocols and tutorials
Chapter 11. Animal Experiments
Blood sampling from animals
Basic skills for animal experiments
Anatomy and physiology of animals
Cancer xenograft animal models
Transgenic animal procedures
Transgenic cancer models
Animal models for depression-like and anxiety-like behavior
Chapter 12. Worm: C. Elegans
C. Elegans Protocols
Chapter 13. HPLC and TLC
HPLC protocols
TLC
Chapter 14. Buffers and Solutions in Lab.
Commonly Used Buffer and solution (114 formats in alphabeta)
Compound solutions (Practical Molecular Biology)How to Make Simple Solutions and Dilutions (Department of Biology, Bates College)Reto's Buffers&Media Book (Private Page)Salt solutions(Practical Molecular Biology)Single Component Solutions (Practical Molecular Biology)Buffers (Gerard R. Lazo)
Antibiotics (Gerard R. Lazo)Buffer Listing (Salmon Lab.)
Cell Culture Media and Solutions (Donis-Keller lab)
Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )
Commonly used solutions (2)--- 1 ) 2 ) 3 )
Solutions for molecular cloning---1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )
Chapter 15. Other Resources
Free eBooks at Library Online
Cinema Online, Free Movies --(1) (2) (3)
Progresses in Life Science
Progress in Cancer Apoptosis -- (1) (2) (3)
Progress in Gene Therapy --- (1) (2) (3)
Progress in Neuronal Regeneration -- (1) (2) (3)
Progress in AIDS Treatment -- (1) (2) (3)
Progress in Organ Transplantation -- (1) (2) (3)
Progress in Stem Cell Research -- (1) (2) (3)
Free eBooks in life science (1)
Free eBooks in life science (2,3)
Most Updated Biomedical Books
Pathway databases
Pathway Search
Biomedical Job Opportunities
Tools for Statistics
Free Software pDRAW32 to Draw DNA Analysis Charts
Biological Educational Resources
Genetics Education
Resources of Medical Biochemistry
Textbooks and Lab Manuals
Copyright: 2008-2009 (c) www.Lab-Manual.Com All Rights Reserved
Contact US: labmanual.com@gmail.com
Dec 31, 2007
Most recent scientific research progress in DNA
- Most recent scientific research progress in DNA
- Hendrik N. Poinar, Carsten Schwarz, Ji Qi, Beth Shapiro, Ross D. E. MacPhee, Bernard Buigues, Alexei Tikhonov, Daniel H. Huson, Lynn P. Tomsho, Alexander Auch, Markus Rampp, Webb Miller, and Stephan C. Schuster
Science 20 January 2006 311: 392-394; published online 20 December 2005 [DOI: 10.1126/science.1123360] (in Reports)
......Paleogenomics: Large-Scale Sequencing of Mammoth DNA Hendrik N. Poinar 1 2 3 * Carsten Schwarz...psu.edu (S.C.S.) 1 McMaster Ancient DNA Center, McMaster University, 1280 Main Street...We sequenced 28 million base pairs of DNA in a metagenomics approach, using a woolly......
Abstract » Full Text » PDF » Supporting Online Material » - Wolfgang Haak, Peter Forster, Barbara Bramanti, Shuichi Matsumura, Guido Brandt, Marc Tänzer, Richard Villems, Colin Renfrew, Detlef Gronenborn, Kurt Werner Alt, and Joachim Burger
Science 11 November 2005 310: 1016-1018 [DOI: 10.1126/science.1118725] (in Reports)
......Report Reports ANTHRO Ancient DNA from the First European Farmers in 7500...Here we present an analysis of ancient DNA from early European farmers. We successfully...stretches of maternally inherited mitochondrial DNA (mtDNA) from 24 out of 57 Neolithic skeletons......
Abstract » Full Text » PDF » Supporting Online Material » - Jennifer A. Leonard, Robert K. Wayne, Jane Wheeler, Raúl Valadez, Sonia Guillén, and Carles Vilà
Science 22 November 2002 298: 1613-1616 [DOI: 10.1126/science.1076980] (in Reports)
...Reports EVOLUTION Ancient DNA Evidence for Old World Origin of New World...DC 20008-0551, USA. Mitochondrial DNA sequences isolated from ancient dog remains...them (13, 14). Consequently, we extracted DNA from bones of 37 dog specimens from archaeological......
Abstract » Full Text » PDF » Supporting Online Material » - D. M. Lambert, P. A. Ritchie, C. D. Millar, B. Holland, A. J. Drummond, and C. Baroni
Science 22 March 2002 295: 2270-2273 [DOI: 10.1126/science.1068105] (in Reports)
......EVOLUTION Rates of Evolution in Ancient DNA from Ad e a lie Penguins D. M...harbor some of the best-preserved ancient DNA yet discovered. From 96 radiocarbon-aged...380 living birds sampled. We demonstrate DNA sequence evolution through time and estimate......
Abstract » Full Text » PDF » Supplemental Data » - James P. Noonan, Graham Coop, Sridhar Kudaravalli, Doug Smith, Johannes Krause, Joe Alessi, Feng Chen, Darren Platt, Svante Pääbo, Jonathan K. Pritchard, and Edward M. Rubin
Science 17 November 2006 314: 1113-1118 [DOI: 10.1126/science.1131412] (in Research Articles)
......Sequencing and Analysis of Neanderthal Genomic DNA James P. Noonan 1 2 Graham Coop 3...targeted method for recovering specific ancient DNA sequences from metagenomic libraries. This...advances in metagenomic analysis of complex DNA mixtures now provide a strategy to recover......
Abstract » Full Text » PDF » Supporting Online Material » - Elizabeth Pennisi
Science 22 March 2002 295: 2197 [DOI: 10.1126/science.295.5563.2197] (in News of the Week)
......-week EVOLUTION EVOLUTIONARY BIOLOGY: Ancient DNA Untangles Evolutionary Paths Elizabeth Pennisi Evolutionary biology. Ancient DNA untangles evolutionary paths. | Comment | News | 0 DNA, Mitochondrial | Science. 2002 Mar 22;295(5563......
Summary » Full Text » PDF » - Hendrik N. Poinar, Matthias Höss, Jeffrey L. Bada, and Svante Pääbo
Science 10 May 1996 272: 864-866 [DOI: 10.1126/science.272.5263.864] (in Reports)
......racemization and the preservation of ancient DNA. | The extent of racemization of aspartic...ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological......
Abstract » References » PDF » - LINDA A. RAUBESON and ROBERT K. JANSEN
Science 27 March 1992 255: 1697-1699 [DOI: 10.1126/science.255.5052.1697] (in Articles)
......Science ARTICLE ARTICLE Chloroplast DNA Evidence on the Ancient Evolutionary Split...vascular plant evolution. The chloroplast DNA (cpDNA) gene order is known to contain an...early vascular land plants. Chloroplast DNA Evidence on the Ancient Evolutionary Split......
Abstract » References » PDF » - Richard D. Wood, Michael Mitchell, John Sgouros, and Tomas Lindahl
Science 16 February 2001 291: 1284-1289 [DOI: 10.1126/science.1056154] (in Reports)
...Reports MOLEC BIOL Human DNA Repair Genes Richard D. Wood Michael...Pittsburgh, PA 15261, USA. Cellular DNA is subjected to continual attack, both...pathways of repair, and 130 known human DNA repair genes are described here. Notable......
Abstract » Full Text » PDF » Supplemental Data » - Tomas Lindahl and Richard D. Wood
Science 3 December 1999 286: 1897-1905 [DOI: 10.1126/science.286.5446.1897] (in Review)
...Quality Control by DNA Repair Tomas Lindahl and...crucial to the individual and to species. DNA damage arises from both endogenous sources...that counteract the mutagenic effects of DNA lesions. This serves to maintain the integrity......
Abstract » Full Text » PDF »
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