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Emergence of DNA Polymerase E Antimutators That Escape Error-Induced Extinction in Yeast
INVESTIGATION Emergence of DNA Polymerase e Antimutators That Escape Error-Induced Extinction in Yeast Lindsey N. Williams, Alan J. Herr, and Bradley D. Preston1 Department of Pathology, University of Washington, Seattle, Washington 98195 ABSTRACT DNA polymerases (Pols) e and d perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol d proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol e proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2D, mlh1D, msh3D msh6D,orpms1D mlh3D) but not with partial MMR loss (msh3D, msh6D, pms1D,ormlh3D), indicating that high levels of unrepaired Pol e errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2D eex mutants encoded second-site changes in Pol e that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol e and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. -
Mutations That Separate the Functions of the Proofreading Subunit of the Escherichia Coli Replicase
G3: Genes|Genomes|Genetics Early Online, published on April 15, 2015 as doi:10.1534/g3.115.017285 Mutations that separate the functions of the proofreading subunit of the Escherichia coli replicase Zakiya Whatley*,1 and Kenneth N Kreuzer*§ *University Program in Genetics & Genomics, Duke University, Durham, NC 27705 §Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 1 © The Author(s) 2013. Published by the Genetics Society of America. Running title: E. coli dnaQ separation of function mutants Keywords: DNA polymerase, epsilon subunit, linker‐scanning mutagenesis, mutation rate, SOS response Corresponding author: Kenneth N Kreuzer, Department of Biochemistry, Box 3711, Nanaline Duke Building, Research Drive, Duke University Medical Center, Durham, NC 27710 Phone: 919 684 6466 FAX: 919 684 6525 Email: [email protected] 1 Present address: Department of Biology, 300 N Washington Street, McCreary Hall, Campus Box 392, Gettysburg College, Gettysburg, PA 17325 Phone: 717 337 6160 Fax: 7171 337 6157 Email: [email protected] 2 ABSTRACT The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3’ 5’ exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our lab identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response following quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. -
The Architecture of a Eukaryotic Replisome
The Architecture of a Eukaryotic Replisome Jingchuan Sun1,2, Yi Shi3, Roxana E. Georgescu3,4, Zuanning Yuan1,2, Brian T. Chait3, Huilin Li*1,2, Michael E. O’Donnell*3,4 1 Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA 2 Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York, USA. 3 The Rockefeller University, 1230 York Avenue, New York, New York, USA. 4 Howard Hughes Medical Institute *Correspondence and requests for materials should be addressed to M.O.D. ([email protected]) or H.L. ([email protected]) ABSTRACT At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase leads the way in front to unwind DNA, and that DNA polymerases (Pol) trail behind the helicase. Here we use single particle electron microscopy to directly image a replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, while Ctf4 and the lagging strand Pol α-primase (Pol α) are behind the helicase. This unexpected architecture indicates that the leading strand DNA travels a long distance before reaching Pol ε, it first threads through the Mcm2-7 ring, then makes a U-turn at the bottom to reach Pol ε at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture, and provides a basis for further structural and biochemical replisome studies. INTRODUCTION DNA is replicated by a multi-protein machinery referred to as a replisome 1,2. Replisomes contain a helicase to unwind DNA, DNA polymerases that synthesize the leading and lagging strands, and a primase that makes short primed sites to initiate DNA synthesis on both strands. -
Orpl, a Member of the Cdcl8/Cdc6 Family of S-Phase Regulators, Is Homologous to a Component of the Origin Recognition Complex M
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12475-12479, December 1995 Genetics Orpl, a member of the Cdcl8/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex M. MuzI-FALCONI AND THOMAS J. KELLY Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Thomas J. Kelly, September 11, 1995 ABSTRACT cdc18+ of Schizosaccharomyces pombe is a is the cdcJ8+ gene (1, 15). Expression of cdcJ8+ from a periodically expressed gene that is required for entry into S heterologous promoter is sufficient to rescue the lethality of a phase and for the coordination of S phase with mitosis. cdc18+ conditional temperature-sensitive (ts) cdc O's mutant. The is related to the Saccharomyces cerevisiae gene CDC6, which has cdcJ8+ gene product, a 65-kDa protein, is essential for the also been implicated in the control of DNA replication. We GI/S transition. Moreover, p65cdclS is a highly labile protein have identified a new Sch. pombe gene, orpl1, that encodes an whose expression is confined to a narrow window at the G,/S 80-kDa protein with amino acid sequence motifs conserved in boundary (unpublished data). These properties are consistent the Cdc18 and Cdc6 proteins. Genetic analysis indicates that with the hypothesis that Cdc18 may play an important role in orpi + is essential for viability. Germinating spores lacking the regulating the initiation of DNA replication at S phase. The orpl + gene are capable of undergoing one or more rounds of Cdc18 protein is homologous to the budding yeast Cdc6 DNA replication but fail to progress further, arresting as long protein, which may have a similar function (16). -
Derepression of Htert Gene Expression Promotes Escape from Oncogene-Induced Cellular Senescence
Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence Priyanka L. Patela, Anitha Surama, Neena Miranib, Oliver Bischofc,d, and Utz Herbiga,e,1 aNew Jersey Medical School-Cancer Center, Rutgers Biomedical and Health Sciences, Newark, NJ 07103; bDepartment of Pathology and Laboratory Medicine, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ 07103; cNuclear Organization and Oncogenesis Unit, Department of Cell Biology and Infection, Institut Pasteur, 75015 Paris, France; dINSERM U993, F-75015 Paris, France; and eDepartment of Microbiology, Biochemistry, and Molecular Genetics, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103 Edited by Victoria Lundblad, Salk Institute for Biological Studies, La Jolla, CA, and approved June 27, 2016 (received for review February 11, 2016) Oncogene-induced senescence (OIS) is a critical tumor-suppressing that occur primarily at fragile sites. The ensuing DNA damage mechanism that restrains cancer progression at premalignant stages, response (DDR) triggers OIS, thereby arresting cells within a few in part by causing telomere dysfunction. Currently it is unknown cell-division cycles after oncogene expression (8, 9). Although most whether this proliferative arrest presents a stable and therefore DSBs in arrested cells are eventually resolved by cellular DSB irreversible barrier to cancer progression. Here we demonstrate that repair processes, some persist and consequently convert the other- cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, wise transient DDR into a more permanent growth arrest. We and after a prolonged period in the senescence arrested state. Cells that others have demonstrated that the persistent DDR is primarily had escaped senescence displayed high oncogene expression levels, telomeric, triggered by irreparable telomeric DSBs (1, 10, 11). -
SUPPLEMENTARY INFORMATION in Silico Signature Prediction
SUPPLEMENTARY INFORMATION In Silico Signature Prediction Modeling in Cytolethal Distending Toxin-Producing Escherichia coli Strains Maryam Javadi, Mana Oloomi*, Saeid Bouzari Department of Molecular Biology, Pasteur Institute of Iran, Tehran 13164, Iran http://www.genominfo.org/src/sm/gni-15-69-s001.pdf Supplementary Table 6. Aalphabetic abbreviation and description of putative conserved domains Alphabetic Abbreviation Description 17 Large terminase protein 2_A_01_02 Multidrug resistance protein 2A0115 Benzoate transport; [Transport and binding proteins, Carbohydrates, organic alcohols] 52 DNA topisomerase II medium subunit; Provisional AAA_13 AAA domain; This family of domains contain a P-loop motif AAA_15 AAA ATPase domain; This family of domains contain a P-loop motif AAA_21 AAA domain AAA_23 AAA domain ABC_RecF ATP-binding cassette domain of RecF; RecF is a recombinational DNA repair ATPase ABC_SMC_barmotin ATP-binding cassette domain of barmotin, a member of the SMC protein family AcCoA-C-Actrans Acetyl-CoA acetyltransferases AHBA_syn 3-Amino-5-hydroxybenzoic acid synthase family (AHBA_syn) AidA Type V secretory pathway, adhesin AidA [Cell envelope biogenesis] Ail_Lom Enterobacterial Ail/Lom protein; This family consists of several bacterial and phage Ail_Lom proteins AIP3 Actin interacting protein 3; Aip3p/Bud6p is a regulator of cell and cytoskeletal polarity Aldose_epim_Ec_YphB Aldose 1-epimerase, similar to Escherichia coli YphB AlpA Predicted transcriptional regulator [Transcription] AntA AntA/AntB antirepressor AraC AraC-type -
DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine
Annu. Rev. Biochem. 1995.64:171-200 Copyright Ii) 1995 byAnnual Reviews Inc. All rights reserved DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine Zvi Kelman and Mike O'Donnell} Microbiology Department and Hearst Research Foundation. Cornell University Medical College. 1300York Avenue. New York. NY }0021 KEY WORDS: DNA replication. multis ubuni t complexes. protein-DNA interaction. DNA-de penden t ATPase . DNA sliding clamps CONTENTS INTRODUCTION........................................................ 172 THE HOLO EN ZYM E PARTICL E. .......................................... 173 THE CORE POLYMERASE ............................................... 175 THE � DNA SLIDING CLAM P............... ... ......... .................. 176 THE yC OMPLEX MATCHMAKER......................................... 179 Role of ATP . .... .............. ...... ......... ..... ............ ... 179 Interaction of y Complex with SSB Protein .................. ............... 181 Meclwnism of the yComplex Clamp Loader ................................ 181 Access provided by Rockefeller University on 08/07/15. For personal use only. THE 't SUBUNIT . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 182 Annu. Rev. Biochem. 1995.64:171-200. Downloaded from www.annualreviews.org AS YMMETRIC STRUC TURE OF HOLO EN ZYM E . 182 DNA PO LYM ER AS E III HOLO ENZ YME AS A REPLIC ATING MACHINE ....... 186 Exclwnge of � from yComplex to Core .................................... 186 Cycling of Holoenzyme on the LaggingStrand -
Gene Disruption of a G4-DNA-Dependent Nuclease In
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6002-6006, June 1995 Genetics Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening (guanine-quartet/KEMI/SEPI/checkpoint/meiosis) ZHIPING Liu, ARNOLD LEE, AND WALTER GILBERT Department of Molecular and Cellular Biology, The Biological Laboratories, 16 Divinity Avenue, Harvard University, Cambridge, MA 02138-2092 Contributed by Walter Gilbert, April 3, 1995 ABSTRACT The yeast gene KEMI (also named (11). A highly conserved feature is that one of the strands is SEPI/DST2/XRNI/RAR5) produces a G4-DNA-dependent very G-rich and always exists as a 3' overhang at the end of the nuclease that binds to G4 tetraplex DNA structure and cuts in chromosome. Telomeres carry out two essential functions. a single-stranded region 5' to the G4 structure. G4-DNA They protect, or stabilize, the ends of linear chromosomes, generated from yeast telomeric oligonucleotides competitively since artificially generated ends (by means of mechanical inhibits the cleavage reaction, suggesting that this enzyme sheering, x-ray irradiation, or enzymatic cleavage) are very may interact with yeast telomeres in vivo. Homozygous dele- unstable (12-14). Furthermore, they serve to circumvent the tions ofthe KEMI gene in yeast block meiosis at the pachytene incomplete DNA replication at the ends of linear chromo- stage, which is consistent with the hypothesis that G4 tetra- somes (15) by extending the G-rich strand through a de novo plex DNA may be involved in homologous chromosome pairing synthesis catalyzed by telomerase to counterbalance the se- during meiosis. We conjectured that the mitotic defects of quence loss at an end in lagging-strand replication (16, 17). -
Functional Characterization of the DNA Polymerase Epsilon and Its Involvement in the Maintenance of Genome Integrity in Arabidopsis Jose Antonio Pedroza-Garcia
Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Jose Antonio Pedroza-Garcia To cite this version: Jose Antonio Pedroza-Garcia. Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis. Plants genetics. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS248. tel-03092326 HAL Id: tel-03092326 https://tel.archives-ouvertes.fr/tel-03092326 Submitted on 2 Jan 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2016SACLS248 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY, préparée à l’Université Paris-Sud ÉCOLE DOCTORALE N° 567 Sciences du Végétal : du Gène à l’Ecosystème Spécialité de doctorat: Biologie Par M. José Antonio Pedroza-Garcia Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Thèse présentée et soutenue à Orsay, le 22 septembre 2016 : Composition du Jury : M. Frugier, Florian Directeur de Recherche, CNRS Président Mme Gallego, Maria Professeure, Université Blaise Pascal Rapporteur M. Bendahmane, Mohammed Directeur de Recherche, INRA Rapporteur Mme Mézard, Christine Directrice de Recherche, CNRS Examinatrice Mme Chabouté, Marie-Edith Directrice de Recherche, CNRS Examinatrice Mme Raynaud, Cécile Chargée de Recherche, CNRS Directrice de thèse Acknowledgments First, I would like to express my huge gratitude to my supervisor, Cécile Raynaud. -
Distinct Co-Evolution Patterns of Genes Associated to DNA Polymerase III Dnae and Polc Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3*
Engelen et al. BMC Genomics 2012, 13:69 http://www.biomedcentral.com/1471-2164/13/69 RESEARCHARTICLE Open Access Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3* Abstract Background: Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results: Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co- evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion: DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world. -
The Molecular Coupling Between Substrate Recognition and ATP Turnover in A
bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader Neha Puri1,2, Amy J. Fernandez1, Valerie L. O’Shea Murray1,3, Sarah McMillan4, James L. Keck4, James M. Berger1,* 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205 2Bristol Myers Squibb, 38 Jackson Road, Devens, MA 01434 3Saul Ewing Arnstein & Lehr, LLP, Centre Square West, 1500 Market Street, 38th Floor, Philadelphia, PA 19102 4Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706 *Corresponding author Email: [email protected] Keywords: DNA replication, AAA+ ATPase, Helicase, Meier-Gorlin Syndrome 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. -
Conversion of OX174 and Fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia Coli (Dnac, Dnad, and Dnag Gene Products/DNA Polymerase III) REED B
Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3233-3237, November 1972 Conversion of OX174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli (dnaC, dnaD, and dnaG gene products/DNA polymerase III) REED B. WICKNER, MICHEL WRIGHT, SUE WICKNER, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Harry Eagle, August 28, 1972 ABSTRACT 4X174 and M13 (fd) single-stranded cir- MATERIALS AND METHODS cular DNAs are converted to their replicative forms by ex- tracts of E. coli pol Al cells. We find that the qX174 DNA- [a-32P]dTTP was obtained from New England Nuclear Corp. dependent reaction requires Mg++, ATP, and all four de- OX174 DNA was prepared by the method of Sinsheimer (7) oxynucleoside triphosphates, but not CTP, UTP, or GTP. or Franke and Ray (8), while fd viral DNA was prepared as This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, described (9). Pancreatic RNase was the highest grade ob- but not that of dnaF (ribonucleotide reductase). The in tainable from Worthington Biochemical Corp. It was further vitro conversion of fd single-stranded DNA to the replica- freed of possible contaminating DNase by heating a solution tive form requires all four ribonucleoside triphosphates, (2 mg/ml in 15 mM sodium citrate, pH 5) at 800 for 10 min. Mg++, and all four deoxynucleoside triphosphates. The E. protein was purified from E. coli strain reaction involves the product of gene dnaE but not those coli unwinding of genes dnaC, dnaD, dnaF, or dnaG.