DOCSLIB.ORG

• Prokaryotic DNA Replication

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
• Prokaryotic DNA Replication • Prokaryotic DNA Replication DNA replication is perfomed by a multienzyme complex >1 MDa DNA Nucleotides Replisome: DNA polymerases Helicase Primase SSBs DNA ligase Clamps (Topoisomerases) 1 Replication is semiconservative, accurrate and fast Accuracy 1 error in 1 billion bases Speed 500 nt/s in bacteria 50 nt/s in mammals Each original strand functions as template for DNA synthesis 2 After each replication cycle, DNA is doubled DNA is synthesized in 5´to 3´direction 3 Polymerisation in detail (dNMP)n + dNTP (dNTP)n+1 + PPi DNA 2 Pi Complementary basepairing and matching hydrogen bonds is required Incorrect basepairing 4 DNA is synthesized by DNA polymerase DNA polymerase III is a protein complex Subunit function not known 3’ exonuclease polymerase clamp dimerisation clamp loader 5 E. coli contains multiple DNA polymerases DNA pol I DNA pol II DNA pol III Number/cell 400 100 10 Speed (nt/s) 16-20 2-5 250-1000 3´exonuclease Yes Yes No 5´exonuclease Yes No No Processivity 3-200 10 000 500 000 Role DNA repair DNA repair Replication RNA primer removal DNA polymerase I Found by Arthur Kornberg, mid 1950’s Three enzymatic activities: • Polymerase activity • 3’ to 5’ exonuclease activity • 5’ to 3’ exonuclease activity Klenow enzyme is lacking one subunit responsible for the 5’ to 3’ exonuclease activity 6 DNA polymerase requires 1. A free 3’-OH group supplied by RNA Primer for start of polymerisation 2. Mg2+ ions for activity in active site 3. A template to copy DNA replication initate at origin of replication Bacterial chromosome doubles in 40 min 7 DNA replication is bidirectional The replication origin OriC in E.coli 245 base pairs AT-rich Initiation proteins bind to 9 bp consensus sequence 8 Inititation of replication at the replication origin Regulation of initiation of replication 9 DNA is synthesized in the replication fork in 5’ to 3’ direction Leading strand synthesis is continuous whereas lagging strand is synthesized in fragments Length of Okazaki fragments in prokaryotes are 1000-2000 nt, in eukaryotes 100-200 nt 10 Mistakes during DNA synthesis are edited This results in a very low error rate of 1 in 1 billion nucleotides 3’ to 5’ exonuclease activity corrects errors 11 Requirements for proofreading mechanism • Addition of nucleotides to RNA primer • Absolute requirement for a match at the 3’ end of the extended strand • 3’ to 5’ exonuclease activity of DNA polymerase • Template DNA is identified by methylation (E. coli) or absence of nicks (eukaryotes) 5’ to 3’ exonuclease activity causes strand displacement/nick translation No net synthesis 12 Helicase unzips double-helix Single strand binding proteins keep strands single stranded Each SSB bind to 7-10 nt Bind in clusters Cooperative binding Lowers Tm of template 13 Binding of SSBs to DNA DNA pol. is attached to strand by Clamp loader and Sliding clamp 14 Sliding clamp Accounts for high processivity: Limits association and dissociation 15 DNA primase Makes the 10 nt RNA primer required for start of replication In beginning of each Okazaki- Fragment RNA primer is later erased and replaced with DNA by DNA pol I 16 DNA ligase Seals the nicks between Okazaki fragments Requires close and free 3’-OH and 5’-P and proper base-pairing NAD+ required in prokaryotes ATP required in eukaryotes Nick sealing by DNA ligase 17 Topoisomerases Relieves torsional stress caused by rotation of DNA ahead of the fork 10 nucleotides = 1 turn Topoisomerase I Breaks one strand of the duplex 18 Mechanism of topoisomerase I 19 Topoisomerase II (DNA gyrase) Breaks both strands of the duplex Introduces negative superhelices ATP dependent 20 Summary of replication DNA is bent duing replication process 21 DNA is proofread during the process Termination of replication The two replication forks are synchronized by 10 23 bp Ter sequences that bind Tus proteins Tus proteins can only be displaced by replisomes coming from one direction 22 Resolvation of replication products by decatenation • Eukaryotic DNA Replication 23 Eukaryotes has some special features Larger genome Multiple linear chromosomes Centromers Telomeres Histones DNA replication DNA replication takes place during the S phase part of the interphase of the cell cycle. S for synthesis. Two identical copies of the chromosome are produced, attached at the centromer. 24 Parts on the yeast chromosome contain Autonomous Replicating Sequence Eukaryotes also contain multiple DNA polymerases DNA pol DNA pol DNA pol DNA pol DNA pol 3´exonuclease No No Yes Yes Yes Fidelity 10-4 -10-5 5x10-4 10-5 10-5 -10-6 10-6 -10-7 Processivity Moderate Low High High High Role Lagging DNA repair Mitochondria Lagging Leading strand l DNA strand strand primer replication replication replication synthesis 25 Inititiation of replication in eukaryotes Due to the eukaryotic chromosome size, multiple replication origins are needed • Eukaryotic replication origins are organized in replicons, 20-80 ori/cluster • Replication is initated all through the S phase • Active chromatin replicate early, condensed chromatin replicate late • A replication bubble is formed at each ori, forks moving in both directions • Each ori is only replicated once Histones are synthesized only during S phase and are added as replication proceeds Some histone parts are ”inherited” some are new The spacing of histones every 200 nt might be the reason for the shorter Okazakifragments in eukaryotes and the slower speed of replication 26 New histones are modified Telomerase recognizes the G-rich 3’- end of the chromosome (telomere) 27 Comparison prokaryotic vs eukaryotic replication Prokaryote (E.coli) Eukaryote (Human) # Origins of replication 1 1000-10000 in replicons Speed of replication 500 nt/s 50 nt/s Time for replication 40 min 8 hours Okazaki fragments 1000-2000 nt 100-200 nt Polymerases 3 (5) 5 (10) Chromosomes 1, circular 46, linear Other Telomeres, histones 28 • Reverse transcription Retroviruses are mobile genetic elements 29 RNA-dependent DNA polymerase 30 31.
Load more

Recommended publications
  • (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution Debayan Purkait†A, Debolina Bandyopadhyay†A, and Padmaja P
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.293639; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Vital Insights into Prokaryotic Genome Compaction by Nucleoid-Associated Protein (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution Debayan Purkait†a, Debolina Bandyopadhyay†a, and Padmaja P. Mishra*a aChemical Sciences Division, Saha Institute of Nuclear Physics, India aHomi Bhaba National Institute (HBNI), India †Both the authors have contributed equally. * Corresponding author Abstract Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a ‘Slacked-Dynamic’ state throughout the observation window where many of them have switched between multiple ‘Wobbling States’ in the course of attainment of packaged form. A range of DNA ‘Flexure Angles’ has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes.
    [Show full text]
  • Cell Cycle Regulation by the Bacterial Nucleoid
    Available online at www.sciencedirect.com ScienceDirect Cell cycle regulation by the bacterial nucleoid David William Adams, Ling Juan Wu and Jeff Errington Division site selection presents a fundamental challenge to all terminate in the terminus region (Ter; 1808). Once organisms. Bacterial cells are small and the chromosome chromosome replication and segregation are complete (nucleoid) often fills most of the cell volume. Thus, in order to the cell is ready to divide. In Bacteria this normally occurs maximise fitness and avoid damaging the genetic material, cell by binary fission and in almost all species this is initiated division must be tightly co-ordinated with chromosome by the assembly of the tubulin homologue FtsZ into a replication and segregation. To achieve this, bacteria employ a ring-like structure (‘Z-ring’) at the nascent division site number of different mechanisms to regulate division site (Figure 1) [1]. The Z-ring then functions as a dynamic selection. One such mechanism, termed nucleoid occlusion, platform for assembly of the division machinery [2,3]. Its allows the nucleoid to protect itself by acting as a template for central role in division also allows FtsZ to serve as a nucleoid occlusion factors, which prevent Z-ring assembly over regulatory hub for the majority of regulatory proteins the DNA. These factors are sequence-specific DNA-binding identified to date [2,4]. Nevertheless, the precise ultra- proteins that exploit the precise organisation of the nucleoid, structure of the Z-ring and whether or not it plays a direct allowing them to act as both spatial and temporal regulators of role in force-generation during division remains contro- bacterial cell division.
    [Show full text]
  • Catalytic Domain of Plasmid Pad1 Relaxase Trax Defines a Group Of
    Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases María Victoria Franciaa,1, Don B. Clewellb,c, Fernando de la Cruzd, and Gabriel Moncaliánd aServicio de Microbiología, Hospital Universitario Marqués de Valdecilla e Instituto de Formación e Investigación Marqués de Valdecilla, Santander 39008, Spain; bDepartment of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109; cDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and dDepartamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria–Consejo Superior de Investigaciones Científicas–Sociedad para el Desarrollo Regional de Cantabria, Santander 39011, Spain Edited by Roy Curtiss III, Arizona State University, Tempe, AZ, and approved July 9, 2013 (received for review May 30, 2013) Plasmid pAD1 is a 60-kb conjugative element commonly found in known relaxases show two characteristic sequence motifs, motif I clinical isolates of Enterococcus faecalis. The relaxase TraX and the containing the catalytic Tyr residue (which covalently attaches to primary origin of transfer oriT2 are located close to each other and the 5′ end of the cleaved DNA) and motif III with the His-triad have been shown to be essential for conjugation. The oriT2 site essential for relaxase activity (facilitating the cleavage reaction contains a large inverted repeat (where the nic site is located) by activation of the catalytic Tyr). This His-triad has been used as adjacent to a series of short direct repeats. TraX does not show a relaxase diagnostic signature (12). fi any of the typical relaxase sequence motifs but is the prototype of Plasmid pAD1 relaxase, TraX, was identi ed (9) and shown to oriT2 a unique family of relaxases (MOB ).
    [Show full text]
  • Relaxation Complexes of Plasmids Colel and Cole2:Unique Site of The
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 3854-3857, October 1974 Relaxation Complexes of Plasmids ColEl and ColE2: Unique Site of the Nick in the Open Circular DNA of the Relaxed Complexes (Escherichia coli/supercoiled DNA/endonuclease/restriction enzyme/DNA replication) MICHAEL A. LOVETT, DONALD G. GUINEY, AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, Calif. 92037 Communicated by Stanley L. Miller, June 26, 1974 ABSTRACT The product of the induced relaxation of volved in the production of a single-strand cleavage in the supercoiled DNA-protein relaxation complexes of colicino- initiation of replication and/or conjugal transfer of plasmid genic factors El (ColEl) and E2 (CoIE2) is an open circular DNA molecule with a strand-specific nick. Cleavage of the DNA. An involvement in such processes would most likely open circular DNA of each relaxed complex with the EcoRI require that the relaxation event take place at a unique site restriction endonuclease demonstrates that the single- in the DNA molecule. In this report, evidence will be pre- strand break is at a unique position. The site of the single- sented from studies using the EcoRI restriction endonuclease strand break in the relaxed ColEl complex is approximately the same distance from the EcoRI cleavage site as the that the strand-specific relaxation event takes place at a origin of ColEl DNA replication. unique site on both the ColEl and CoIE2 plasmid molecules. The location of this site, at least in the case of the ColEl Many of extrachromosomal circular DNA elements (plas- plasmid, is approximately the same distance from the single mids) of Escherichia coli, differing in molecular weight, num- EcoRI site as the origin of replication for this plasmid.
    [Show full text]
  • Teacher Notes Science Nspired
    DNA Replication TEACHER NOTES SCIENCE NSPIRED Science Objectives In this lesson, students will • Explore how the structure of DNA supports its semi-conservative replication • Identify the name and function of several key enzymes in DNA replication • Recognize the function of Okazaki fragments in DNA replication Vocabulary TI-Nspire™ Technology Skills: • Double helix • Leading strand • Download a TI-Nspire • Lagging strand • Polymerase I • Polymerase III • Helicase document • Base pairs • Okazaki Fragments • Open a document • Primase • Move between pages • Open Directions Box About the Lesson • Explore ‘Hot Spots’ • Using three simulations, students will interact with DNA replication to explore semi-conservative replication and identify Tech Tips: specific enzymes and their roles in replication. Assessments are Make sure that students know embedded in the activity to engage discussion and gauge how to move between pages by learning. pressing /¡ (left arrow) and • As a result, students will: /¢ (right arrow). • Learn the basic functions of the following DNA replication enzymes: helicase, primase, ligase, polymerase I and III. Lesson Materials: • Learn how the double helix reproduces in a semi- Student Activity • DNA_Replication_Student.do conservative fashion c • Learn the role and function of Okazaki fragments in • DNA_Replication_Student.pd replication of the lagging strand. f TI-Nspire document TI-Nspire™ Navigator™ • DNA_Replication.tns Not required. If using Navigator: • Send out the DNA_Replication.tns file. • Monitor student progress
    [Show full text]
  • DNA Structure and Replication Three Key Features Needed for Any Model
    DNA structure and replication What was known? 1) Hereditary factors were associated with specific traits 2) One-gene-one-protein model - from mapping genes for biosynthetic pathways 3) Genes are on chromosomes 4) Chromosomes are made up of DNA and protein Three key features needed for any model of DNA structure 1) Must allow for faithful replication 2) Must have information content 3) Must be able to change in order to explain mutations 1 Rosalind Franklin James Watson Francis Crick X-ray diffraction Maurice Wilkins Photograph of B-DNA Linus Pauling Nature 171, 737-738 (1953) © Macmillan Publishers Ltd. Molecular structure of Nucleic Acids WATSON, J. D. & CRICK, F. H. C. Features of the double helix A Structure for Deoxyribose Nucleic Acid 1) Two parallel strands We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest………… 2) Bases held together by H-bonds 3) Phosphodiester backbone 4) The base is attached to the position 1 on the sugar 5) Base pair stack - provides Figure 1. This figure is purely diagrammatic. The two ribbons symbolize the two phophate-sugar chains, and the horizonal rods stability the pairs of bases holding the chains together. The vertical line marks the fibre axis. 6) Contains a major and …………….It has not escaped our notice that the specific pairing we minor groove have postulated immediately suggests a possible copying mechanism for the genetic material. Three key features needed for any model of DNA structure
    [Show full text]
  • Processivity of DNA Polymerases: Two Mechanisms, One Goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2
    Minireview 121 Processivity of DNA polymerases: two mechanisms, one goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2 Replicative DNA polymerases are highly processive Processive DNA synthesis by cellular replicases and the enzymes that polymerize thousands of nucleotides without bacteriophage T4 replicase dissociating from the DNA template. The recently Until recently, the only mechanism for high processivity determined structure of the Escherichia coli bacteriophage that was understood in detail was that utilized by cellular T7 DNA polymerase suggests a unique mechanism that replicases and the replicase of bacteriophage T4. This underlies processivity, and this mechanism may generalize mechanism involves a ring-shaped protein called a ‘DNA to other replicative polymerases. sliding clamp’ that encircles the DNA and tethers the polymerase catalytic unit to the DNA [3,4]. The three- Addresses: 1Department of Molecular Biology, Memorial Sloan- dimensional structures of several sliding clamps have been Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, 2 determined: the eukaryotic proliferating cell nuclear USA and Laboratory of DNA Replication, Howard Hughes Medical β Institute, The Rockefeller University, 1230 York Avenue, New York, NY antigen (PCNA) [5,6]; the subunit of the prokaryotic 10021, USA. DNA polymerase III [7]; and the bacteriophage T4 gene 45 protein (gp45) (J Kuriyan, personal communication) *Corresponding author. (Figure 1). The overall structure of these clamps is very E-mail: [email protected] similar; the PCNA, β subunit and gp45 rings are super- Structure 15 February 1998, 6:121–125 imposable [8]. Each ring has similar dimensions and a http://biomednet.com/elecref/0969212600600121 central cavity large enough to accommodate duplex DNA (Figure 1).
    [Show full text]
  • Primer Release Is the Rate-Limiting Event in Lagging-Strand Synthesis
    Primer release is the rate-limiting event in lagging- strand synthesis mediated by the T7 replisome Alfredo J. Hernandeza, Seung-Joo Leea, and Charles C. Richardsona,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, April 18, 2016 (sent for review December 30, 2015; reviewed by Nicholas E. Dixon and I. Robert Lehman) DNA replication occurs semidiscontinuously due to the antiparallel but also for enabling the use of short oligoribonucleotides by T7 DNA strands and polarity of enzymatic DNA synthesis. Although DNA polymerase. Critically, the primase domain also fulfills two the leading strand is synthesized continuously, the lagging strand additional roles apart from primer synthesis: it prevents disso- is synthesized in small segments designated Okazaki fragments. ciation of the extremely short tetramer, stabilizing it with the Lagging-strand synthesis is a complex event requiring repeated template, and it secures it in the polymerase active site (10, 12). cycles of RNA primer synthesis, transfer to the lagging-strand Here we show that the rate-limiting step in initiation of Okazaki polymerase, and extension effected by cooperation between DNA fragments by the T7 replisome is primer release from the primase primase and the lagging-strand polymerase. We examined events domain of gp4. In the absence of gp2.5, an additional step, distinct controlling Okazaki fragment initiation using the bacteriophage from primer release, also limits primer extension. The presence of T7 replication system. Primer utilization by T7 DNA polymerase is gp2.5 promotes efficient primer formation and primer utilization. slower than primer formation.
    [Show full text]
  • Junction Ribonuclease: an Activity in Okazaki Fragment Processing
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2244–2249, March 1998 Biochemistry Junction ribonuclease: An activity in Okazaki fragment processing RICHARD S. MURANTE,LEIGH A. HENRICKSEN, AND ROBERT A. BAMBARA* Department of Biochemistry and Biophysics, and Cancer Center, Box 712, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642 Communicated by I. Robert Lehman, Stanford University School of Medicine, Stanford, CA, December 29, 1997 (received for review September 15, 1997) ABSTRACT The initiator RNAs of mammalian Okazaki and removal of RNA primers. Reconstitution assays with both fragments are thought to be removed by RNase HI and the mouse cell and SV40 replication proteins require RNase HI for 5*-3* flap endonuclease (FEN1). Earlier evidence indicated the maturation of lagging strands in vitro (12, 13). that the cleavage site of RNase HI is 5* of the last ribonucle- Although cleavage of long RNAyDNA hybrids is random, otide at the RNA-DNA junction on an Okazaki substrate. In cleavage of certain substrates is clearly structure specific. Eder current work, highly purified calf RNase HI makes this exact et al. (16) found that when a series of ribonucleotides were cleavage in Okazaki fragments containing mismatches that followed by a 39 DNA segment and annealed to DNA to form distort the hybrid structure of the heteroduplex. Furthermore, a duplex, human RNase HI preferentially cleaved to leave a even fully unannealed Okazaki fragments were cleaved. DNA segment with a 59 monoribonucleotide. In reactions Clearly, the enzyme recognizes the transition from RNA to reconstituting Okazaki fragment processing, RNase HI from DNA on a single-stranded substrate and not the RNAyDNA calf thymus similarly cleaved the RNA-DNA strand 59 to the heteroduplex structure.
    [Show full text]
  • S Na P S H O T: D N a Mism a Tc H R E P a Ir
    SnapShot: Repair DNA Mismatch Scott A. Lujan, and Thomas Kunkel A. Larrea, Andres Park, NC 27709, USA Triangle Health Sciences, NIH, DHHS, Research National Institutes of Environmental 730 Cell 141, May 14, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.05.002 See online version for legend and references. SnapShot: DNA Mismatch Repair Andres A. Larrea, Scott A. Lujan, and Thomas A. Kunkel National Institutes of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA Mismatch Repair in Bacteria and Eukaryotes Mismatch repair in the bacterium Escherichia coli is initiated when a homodimer of MutS binds as an asymmetric clamp to DNA containing a variety of base-base and insertion-deletion mismatches. The MutL homodimer then couples MutS recognition to the signal that distinguishes between the template and nascent DNA strands. In E. coli, the lack of adenine methylation, catalyzed by the DNA adenine methyltransferase (Dam) in newly synthesized GATC sequences, allows E. coli MutH to cleave the nascent strand. The resulting nick is used for mismatch removal involving the UvrD helicase, single-strand DNA-binding protein (SSB), and excision by single-stranded DNA exonucleases from either direction, depending upon the polarity of the nick relative to the mismatch. DNA polymerase III correctly resynthesizes DNA and ligase completes repair. In bacteria lacking Dam/MutH, as in eukaryotes, the signal for strand discrimination is uncertain but may be the DNA ends associated with replication forks. In these bacteria, MutL harbors a nick-dependent endonuclease that creates a nick that can be used for mismatch excision. Eukaryotic mismatch repair is similar, although it involves several dif- ferent MutS and MutL homologs: MutSα (MSH2/MSH6) recognizes single base-base mismatches and 1–2 base insertion/deletions; MutSβ (MSH2/MSH3) recognizes insertion/ deletion mismatches containing two or more extra bases.
    [Show full text]
  • DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer
    cancers Review DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer Youri I. Pavlov 1,2,* , Anna S. Zhuk 3 and Elena I. Stepchenkova 2,4 1 Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Genetics and Biotechnology, Saint-Petersburg State University, 199034 Saint Petersburg, Russia; [email protected] 3 International Laboratory of Computer Technologies, ITMO University, 197101 Saint Petersburg, Russia; [email protected] 4 Laboratory of Mutagenesis and Genetic Toxicology, Vavilov Institute of General Genetics, Saint-Petersburg Branch, Russian Academy of Sciences, 199034 Saint Petersburg, Russia * Correspondence: [email protected] Received: 30 September 2020; Accepted: 13 November 2020; Published: 24 November 2020 Simple Summary: The etiology of cancer is linked to the occurrence of mutations during the reduplication of genetic material. Mutations leading to low replication fidelity are the culprits of many hereditary and sporadic cancers. The archetype of the current model of replication fork was proposed 30 years ago. In the sequel to our 2010 review with the words “years after” in the title inspired by A. Dumas’s novels, we go over new developments in the DNA replication field and analyze how they help elucidate the effects of the genetic variants of DNA polymerases on cancer. Abstract: Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization.
    [Show full text]
  • Effects of Abasic Sites and DNA Single-Strand Breaks on Prokaryotic RNA Polymerases (Apurinic/Apyrimidinic Sites/Transcription/DNA Damage) WEI ZHOU* and PAUL W
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6601-6605, July 1993 Biochemistry Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases (apurinic/apyrimidinic sites/transcription/DNA damage) WEI ZHOU* AND PAUL W. DOETSCH*tt Departments of *Biochemistry and tRadiation Oncology and tDivision of Cancer Biology, Emory University School of Medicine, Atlanta, GA 30322 Communicated by Philip C. Hanawalt, April 19, 1993 ABSTRACT Abasic sites are thought to be the most fre- effects of such damage on both transcription initiation and quently occurring cellular DNA damage and are generated elongation processes (19, 20). We wished to determine the spontaneously or as the result of chemical or radiation damage effects of AP sites on the transcription elongation process by to DNA. In contrast to the wealth of information that exists on using a defined system that would provide direct information the effects of abasic sites on DNA polymerases, very little is regarding the ability of RNA polymerases to bypass or be known about how these lesions interact with RNA polymerases. blocked by AP sites and, if bypass was observed, to deter- An in vitro transcription system was used to determine the mine the nature of the base insertion opposite such lesions. effects of abasic sites and single-strand breaks on transcrip- For these studies, we constructed a DNA template containing tional elongation. DNA templates were constructed containing a single AP site placed at a unique location downstream from single abasic sites or nicks placed at unique locations down- either the SP6 or tac promoter and carried out in vitro stream from two different promoters and were transcribed by transcription experiments with SP6 and E.
    [Show full text]