DOCSLIB.ORG

The Devil Is in the Details for DNA Mismatch Repair COMMENTARY

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Devil Is in the Details for DNA Mismatch Repair COMMENTARY COMMENTARY The Devil is in the details for DNA mismatch repair COMMENTARY Peggy Hsieha,1 and Yongliang Zhanga Ensuring high-fidelity DNA replication is essential a strand-specific signal for excision can be far apart) (7–9). for maintaining genome stability in organisms from MMR in eukaryotes features two families of MMR pro- Escherichia coli to humans. This task requires an intricate teins, heterodimeric homologs of bacterial MutS (MSH) network of cellular components that carries out replica- or MutL (MLH). Early models of E. coli MMR invoke tion and postreplication DNA repair (1). In eukaryotes, MMR protein-mediated DNA looping between a mis- replication is carried out by Pol e and Pol δ that synthesize match and a DNA-methylation excision signal or ATP- the leading and lagging strands, respectively, using short driven MutS translocation along the DNA. The nucleotide RNA–DNA primers synthesized by Pol α. Although repli- switch model posits the existence of multiple diffusing cation gives rise to a low spontaneous mutation rate of MSH–MLH clamps (8). Other studies suggest that a single − ∼10 10 mutations per base pair per generation, errors in MSH clamp at a mismatch can load multiple MLH clamps the nucleotide incorporation step occur about once every to license excision (1, 9). MutSα (MSH2-MSH6) and MutSβ + 104 to 105 insertions on average, although the frequency (MSH2-MSH3) are related to the AAA family of ATPases varies considerably depending on a number of parame- and carry out mismatch recognition (10, 11). MutL homo- ters including the particular mismatch, chromosomal con- logs MutLα (yeast ScMlh1-Pms1 and human HsMLH1- text, and the DNA polymerase. In addition to stringent PMS2), MutLβ (Mlh1-Mlh2), and MutLγ (Mlh1-Mlh3) have selectivity at each step of nucleotide incorporation, high- N- and C-terminal domains connected by a long helix fidelity DNA polymerases possess 3′-endonuclease activ- (12). The N-terminal domain mediates interactions with ities that act as proofreaders, removing errant bases MutS proteins and DNA and has a GHKL family ATP- whose abnormal geometries act as speed bumps, allow- binding motif. The C-terminal dimerization domain has ing excision of the incorrect base and insertion of the a latent endonuclease activity in Pms1 (HsPMS2) and correct one. Working closely with the replication machin- Mlh3 that is activated by proliferating cell nuclear antigen ery is a postreplication DNA repair pathway: DNA mis- (PCNA), a replication processivity clamp. Mismatches be- match repair (MMR). MMR targets replication errors long to two classes, base–base mispairs (e.g., G opposite that have escaped proofreading by excising a region T) and unpaired insertion and deletion (indel) mismatches that contains the mismatched base(s) on the newly at repetitive sequences caused by strand slippage. Indels synthesized strand and giving a high-fidelity DNA are recalcitrant to proofreading because they are often too polymerase a second chance. In cells whose MMR far from the polymerase active site, leading to microsatel- function is compromised by mutation or epigenetic lite instability, the contraction and expansion of mono- silencing a hypermutator phenotype ensues. Loss of and dinucleotide repeats whose presence in MMR- MMR results in inherited cancer susceptibility (e.g., Lynch deficient bacteria and yeast presaged the discovery of syndrome), as well as an increased incidence of sporadic MMR defects in Lynch syndrome colorectal cancer (13). cancers (2). MMR has been reconstituted with a heterodu- In vitro, 5′ MMR requires MutSα or β,EXO1,anobligate5′ plex (mismatched) DNA substrate and purified proteins to 3′ exonuclease, RPA single-strand binding protein, RFC from E. coli and subsequently with yeast and human pro- clamp loader PCNA, and Pol δ or e, whereas 3′ MMR teins (3–5). Until now, eukaryotic systems have all used Pol requires these proteins plus MLH (e.g., MutLα)(3–6). δ. In PNAS, Bowen and Kolodner report the first reconsti- The first stage of MMR is mismatch recognition by tution with purified Saccharomyces cerevisiae proteins of MutSα or MutSβ (Fig. 1A). Using related but distinct mis- 5′ and 3′ MMR that is dependent on Pol e (6). match binding sites, MutSα repairs base–base mispairs and The general schemes of MMR seem deceptively small indels of 1–2 nt, whereas MutSβ handles primarily simple (Fig. 1). The newly synthesized strand containing indels of 1–15 bases. Mismatch provoked ADP → ATP mismatches is targeted for excision followed by high- exchange in the nucleotide binding sites converts MutSα fidelity resynthesis and ligation. Nevertheless, any scheme and MutSβ into sliding clamps that diffuse along the DNA, mustreconcileactionatadistance(i.e.,themismatchand facilitating the mismatch search and subsequent interaction aGenetics & Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Author contributions: P.H. and Y.Z. wrote the paper. The authors declare no conflict of interest. See companion article 10.1073/pnas.1701753114. 1To whom correspondence should be addressed. Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1702747114 PNAS Early Edition | 1of3 Downloaded by guest on September 29, 2021 Fig. 1. Schemes for in vitro eukaryotic MMR. (Left)5′ MMR. (A) The MutS homolog proteins (MSH, purple) MutSα (MSH2-MSH6), or MutSβ (MSH2-MSH3) recognize and bind a mismatch. RPA (green) bound to single-strand DNA prevents EXO1 (blue) from accessing and degrading DNA. (B) In the sliding clamp model, MutSα/β at a mismatch binds ATP (yellow) and undergoes nucleotide switch activation, becoming a sliding clamp that diffuses along the DNA. Multiple MSH clamps are loaded at a single mismatch. The interaction of EXO1 with MSH sliding clamps overcomes the RPA barrier and activates EXO1 for 5′ to 3′ excision from the 5′ nick. MutL homolog proteins (MLH, pink) (MutLα is ScMlh1-Pms1 or HsMLH1-PMS2) bind ATP and may interact with MSH sliding clamps, though MLH is not absolutely required in vitro for 5’ MMR. In other models, MSH remains at the mismatch to authorize excision or can load multiple MLH clamps onto the DNA in the vicinity of the mismatch (not shown). (C) In the sliding clamp model, the EXO1/MSH complex dissociates after excising several hundred nucleotides. Iterative rounds of MSH-EXO1 excision create an excision tract coated with RPA that extends from the 5′ nick to just beyond the mismatch. MLH may limit excision by modulating the number of MSH clamps on DNA. (D) RFC (not shown) loads PCNA clamps (orange) with specific orientation at 3′ termini of strand breaks or gaps, and PCNA facilitates high-fidelity DNA synthesis by Pol δ or e (red). (E) DNA ligase I (green) seals the nick. (Right)3′ MMR. (A) MSH recognizes a mismatch. (B) In the sliding clamp model, ATP-dependent binding and nucleotide switching creates MSH sliding clamps that diffuse from the mismatch. The interaction of ATP-bound MLH heterodimers with MSH sliding clamps and PCNA oriented with respect to 3′ termini activates MLH strand-specific nicking. Alternatively, ATP-activated MSH may remain at the mismatch to load MLH and activate nicking (not shown). (C) Excision is EXO1-dependent or -independent, leading to an RPA-coated excision track. An EXO1-independent Pol δ strand- displacement pathway is not shown. (D)Polδ or e (red) with the aid of PCNA completes gap filling. (E) DNA ligase I (green) seals the nick. with MutLα. PCNA interacts with several MMR components including originate elsewhere. Currently, the leading candidate is PCNA. In MutS and MutL homologs, EXO1, Pol δ,andPole.MSH3and vitro MMR reactions require a preexisting nick or single-strand gap MSH6 interact with PCNA via PCNA-interaction-peptide (PIP) motifs in the heteroduplex DNA substrate that can reside on the 5′ (5′ MMR) in an N-terminal domain. The ability of PCNA to provide a link be- or 3′ (3′ MMR) side of the mismatch. Importantly, this strand discon- tween replication and MMR and to facilitate the mismatch search is tinuity serves to direct excision exclusively to the nicked strand. These supported by the observation that colocalization of ScMutSα foci with nicks and gaps are potential PCNA loading sites. replication centers is dependent on an intact PIP motif in Msh6 (14). In 5′ MMR reactions, excision is catalyzed by EXO1, a 5′ to 3′ Paradoxically, the mutator phenotype of Msh6 PIP mutants is relatively exonuclease that is activated by MutSα in a mismatch-dependent mild, suggesting an alternate pathway for mismatch recognition that manner (Fig. 1, Left). Although HsMLH1-PMS2 is not required for re- does not require interaction with the replication machinery. ScMsh2, pair in vitro, its presence modulates the excision step such that exci- but not Msh6, has been implicated in MSH–MLH interactions, and a sion tracts terminate just past the mismatch (5). Real-time single- cocrystal structure of E. coli MutS cross-linked to the N-terminal do- molecule imaging studies of the excision step of 5′ MMR support a main of MutL indicates extensive conformational changes in the com- dynamic molecular switch/sliding clamp model in which multiple ATP- plex (9, 15). Defining the physical interaction between MSH and MLH bound HsMutSα sliding clamps diffuse along the DNA following mis- proteins and PCNA and determining the dynamics of complex forma- match binding (16). Although HsRPA bound to a single-strand gap or tion and dissociation remain important problems. nick inhibits EXO1, preventing runaway excision, an HsMutSα-EXO1 The next stage is excision, a carefully regulated step whose details complex at the 5′ break can promote excision over a distance of are unresolved (Fig. 1 B and C). A central tenet of MMR is that DNA ∼800 nt. In the case of a mismatch located some distance from the excision must be targeted to the newly synthesized strand containing initiating strand break or gap, iterations of MutSα/EXO1-mediated the error rather than the parental or template strand.
Load more

Recommended publications
  • Glossary - Cellbiology
    1 Glossary - Cellbiology Blotting: (Blot Analysis) Widely used biochemical technique for detecting the presence of specific macromolecules (proteins, mRNAs, or DNA sequences) in a mixture. A sample first is separated on an agarose or polyacrylamide gel usually under denaturing conditions; the separated components are transferred (blotting) to a nitrocellulose sheet, which is exposed to a radiolabeled molecule that specifically binds to the macromolecule of interest, and then subjected to autoradiography. Northern B.: mRNAs are detected with a complementary DNA; Southern B.: DNA restriction fragments are detected with complementary nucleotide sequences; Western B.: Proteins are detected by specific antibodies. Cell: The fundamental unit of living organisms. Cells are bounded by a lipid-containing plasma membrane, containing the central nucleus, and the cytoplasm. Cells are generally capable of independent reproduction. More complex cells like Eukaryotes have various compartments (organelles) where special tasks essential for the survival of the cell take place. Cytoplasm: Viscous contents of a cell that are contained within the plasma membrane but, in eukaryotic cells, outside the nucleus. The part of the cytoplasm not contained in any organelle is called the Cytosol. Cytoskeleton: (Gk. ) Three dimensional network of fibrous elements, allowing precisely regulated movements of cell parts, transport organelles, and help to maintain a cell’s shape. • Actin filament: (Microfilaments) Ubiquitous eukaryotic cytoskeletal proteins (one end is attached to the cell-cortex) of two “twisted“ actin monomers; are important in the structural support and movement of cells. Each actin filament (F-actin) consists of two strands of globular subunits (G-Actin) wrapped around each other to form a polarized unit (high ionic cytoplasm lead to the formation of AF, whereas low ion-concentration disassembles AF).
    [Show full text]
  • Deficiency in the DNA Repair Protein ERCC1 Triggers a Link Between Senescence and Apoptosis in Human Fibroblasts and Mouse Skin
    Lawrence Berkeley National Laboratory Recent Work Title Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin. Permalink https://escholarship.org/uc/item/73j1s4d1 Journal Aging cell, 19(3) ISSN 1474-9718 Authors Kim, Dong Eun Dollé, Martijn ET Vermeij, Wilbert P et al. Publication Date 2020-03-01 DOI 10.1111/acel.13072 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Received: 10 June 2019 | Revised: 7 October 2019 | Accepted: 30 October 2019 DOI: 10.1111/acel.13072 ORIGINAL ARTICLE Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin Dong Eun Kim1 | Martijn E. T. Dollé2 | Wilbert P. Vermeij3,4 | Akos Gyenis5 | Katharina Vogel5 | Jan H. J. Hoeijmakers3,4,5 | Christopher D. Wiley1 | Albert R. Davalos1 | Paul Hasty6 | Pierre-Yves Desprez1 | Judith Campisi1,7 1Buck Institute for Research on Aging, Novato, CA, USA Abstract 2Centre for Health Protection Research, ERCC1 (excision repair cross complementing-group 1) is a mammalian endonuclease National Institute of Public Health and that incises the damaged strand of DNA during nucleotide excision repair and inter- the Environment (RIVM), Bilthoven, The −/Δ Netherlands strand cross-link repair. Ercc1 mice, carrying one null and one hypomorphic Ercc1 3Department of Molecular Genetics, allele, have been widely used to study aging due to accelerated aging phenotypes Erasmus University Medical Center, −/Δ Rotterdam, The Netherlands in numerous organs and their shortened lifespan. Ercc1 mice display combined 4Princess Máxima Center for Pediatric features of human progeroid and cancer-prone syndromes.
    [Show full text]
  • Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases
    cells Review Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases Paulina Prorok 1 , Inga R. Grin 2,3, Bakhyt T. Matkarimov 4, Alexander A. Ishchenko 5 , Jacques Laval 5, Dmitry O. Zharkov 2,3,* and Murat Saparbaev 5,* 1 Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany; [email protected] 2 SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., 630090 Novosibirsk, Russia; [email protected] 3 Center for Advanced Biomedical Research, Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., 630090 Novosibirsk, Russia 4 National Laboratory Astana, Nazarbayev University, Nur-Sultan 010000, Kazakhstan; [email protected] 5 Groupe «Mechanisms of DNA Repair and Carcinogenesis», Equipe Labellisée LIGUE 2016, CNRS UMR9019, Université Paris-Saclay, Gustave Roussy Cancer Campus, F-94805 Villejuif, France; [email protected] (A.A.I.); [email protected] (J.L.) * Correspondence: [email protected] (D.O.Z.); [email protected] (M.S.); Tel.: +7-(383)-3635187 (D.O.Z.); +33-(1)-42115404 (M.S.) Abstract: It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the Citation: Prorok, P.; Grin, I.R.; major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field Matkarimov, B.T.; Ishchenko, A.A.; and alkylating metabolic radicals added to these threats.
    [Show full text]
  • Reconstitution of Long and Short Patch Mismatch Repair Reactions Using Saccharomyces Cerevisiae Proteins
    Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins Nikki Bowena, Catherine E. Smitha, Anjana Srivatsana, Smaranda Willcoxb,c, Jack D. Griffithb,c, and Richard D. Kolodnera,d,e,f,g,1 aLudwig Institute for Cancer Research, Departments of dMedicine and eCellular and Molecular Medicine, fMoores-University of California, San Diego Cancer Center, and gInstitute of Genomic Medicine, University of California San Diego School of Medicine, La Jolla, CA 92093; and bLineberger Cancer Center and cDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 Contributed by Richard D. Kolodner, October 8, 2013 (sent for review September 9, 2013) A problem in understanding eukaryotic DNA mismatch repair In eukaryotic MMR, mispairs are bound by MutS homolog 2 (MMR) mechanisms is linking insights into MMR mechanisms from (Msh2)–MutS homolog 6 (Msh6) and Msh2–MutS homolog 3 genetics and cell-biology studies with those from biochemical (Msh3), two partially redundant complexes of MutS-related pro- studies of MMR proteins and reconstituted MMR reactions. This teins (3, 4, 18, 19). These complexes recruit a MutL-related type of analysis has proven difficult because reconstitution ap- complex, called MutL homoloh 1 (Mlh1)–postmeiotic segrega- proaches have been most successful for human MMR whereas tion 1 (Pms1) in S. cerevisiae and Mlh1–postmeiotic segregation – – analysis of MMR in vivo has been most advanced in the yeast 2 (Pms2) in human and mouse (3, 4, 20 23). The Mlh1 Pms1/ Saccharomyces cerevisiae. Here, we describe the reconstitution of Pms2 complex has an endonuclease activity suggested to play MMR reactions using purified S.
    [Show full text]
  • Paul Modrich Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA
    Mechanisms in E. coli and Human Mismatch Repair Nobel Lecture, December 8, 2015 by Paul Modrich Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA. he idea that mismatched base pairs occur in cells and that such lesions trig- T ger their own repair was suggested 50 years ago by Robin Holliday in the context of genetic recombination [1]. Breakage and rejoining of DNA helices was known to occur during this process [2], with precision of rejoining attributed to formation of a heteroduplex joint, a region of helix where the two strands are derived from the diferent recombining partners. Holliday pointed out that if this heteroduplex region should span a genetic diference between the two DNAs, then it will contain one or more mismatched base pairs. He invoked processing of such mismatches to explain the recombination-associated phenomenon of gene conversion [1], noting that “If there are enzymes which can repair points of damage in DNA, it would seem possible that the same enzymes could recognize the abnormality of base pairing, and by exchange reactions rectify this.” Direct evidence that mismatches provoke a repair reaction was provided by bacterial transformation experiments [3–5], and our interest in this efect was prompted by the Escherichia coli (E. coli) work done in Matt Meselson’s lab at Harvard. Using artifcially constructed heteroduplex DNAs containing multiple mismatched base pairs, Wagner and Meselson [6] demonstrated that mismatches elicit a repair reaction upon introduction into the E. coli cell. Tey also showed that closely spaced mismatches, mismatches separated by a 1000 base pairs or so, are usually repaired on the same DNA strand.
    [Show full text]
  • (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]
  • Table 2. Significant
    Table 2. Significant (Q < 0.05 and |d | > 0.5) transcripts from the meta-analysis Gene Chr Mb Gene Name Affy ProbeSet cDNA_IDs d HAP/LAP d HAP/LAP d d IS Average d Ztest P values Q-value Symbol ID (study #5) 1 2 STS B2m 2 122 beta-2 microglobulin 1452428_a_at AI848245 1.75334941 4 3.2 4 3.2316485 1.07398E-09 5.69E-08 Man2b1 8 84.4 mannosidase 2, alpha B1 1416340_a_at H4049B01 3.75722111 3.87309653 2.1 1.6 2.84852656 5.32443E-07 1.58E-05 1110032A03Rik 9 50.9 RIKEN cDNA 1110032A03 gene 1417211_a_at H4035E05 4 1.66015788 4 1.7 2.82772795 2.94266E-05 0.000527 NA 9 48.5 --- 1456111_at 3.43701477 1.85785922 4 2 2.8237185 9.97969E-08 3.48E-06 Scn4b 9 45.3 Sodium channel, type IV, beta 1434008_at AI844796 3.79536664 1.63774235 3.3 2.3 2.75319499 1.48057E-08 6.21E-07 polypeptide Gadd45gip1 8 84.1 RIKEN cDNA 2310040G17 gene 1417619_at 4 3.38875643 1.4 2 2.69163229 8.84279E-06 0.0001904 BC056474 15 12.1 Mus musculus cDNA clone 1424117_at H3030A06 3.95752801 2.42838452 1.9 2.2 2.62132809 1.3344E-08 5.66E-07 MGC:67360 IMAGE:6823629, complete cds NA 4 153 guanine nucleotide binding protein, 1454696_at -3.46081884 -4 -1.3 -1.6 -2.6026947 8.58458E-05 0.0012617 beta 1 Gnb1 4 153 guanine nucleotide binding protein, 1417432_a_at H3094D02 -3.13334396 -4 -1.6 -1.7 -2.5946297 1.04542E-05 0.0002202 beta 1 Gadd45gip1 8 84.1 RAD23a homolog (S.
    [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]
  • Phosphate Steering by Flap Endonuclease 1 Promotes 50-flap Specificity and Incision to Prevent Genome Instability
    ARTICLE Received 18 Jan 2017 | Accepted 5 May 2017 | Published 27 Jun 2017 DOI: 10.1038/ncomms15855 OPEN Phosphate steering by Flap Endonuclease 1 promotes 50-flap specificity and incision to prevent genome instability Susan E. Tsutakawa1,*, Mark J. Thompson2,*, Andrew S. Arvai3,*, Alexander J. Neil4,*, Steven J. Shaw2, Sana I. Algasaier2, Jane C. Kim4, L. David Finger2, Emma Jardine2, Victoria J.B. Gotham2, Altaf H. Sarker5, Mai Z. Her1, Fahad Rashid6, Samir M. Hamdan6, Sergei M. Mirkin4, Jane A. Grasby2 & John A. Tainer1,7 DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability. 1 Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
    [Show full text]
  • Chromosome Replication Duringmeiosis
    Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3087-3091, November 1973 Chromosome Replication During Meiosis: Identification of Gene Functions Required for Premeiotic DNA Synthesis (yeast) ROBERT ROTH Biology Department, Illinois Institute of Technology, Chicago, Ill. 60616 Communicated by Herschel L. Roman, May 29, 1973 ABSTRACT Recent comparisons of chromosome repli- tained provide additional evidence that distinct biochemical cation in meiotic and mitotic cells have revealed signifi- reactions do distinguish the last premeiotic replication from cant differences in both the rate and pattern of DNA synthesis during the final duplication preceding meiosis. replication during growth. These differences suggested that unique gene functions might be required for premeiotic replication that were not MATERIALS AND METHODS necessary for replication during growth. To provide Yeast Strains. Mutants M10-2B and M10-6A were isolated evidence for such functions, we isolated stage-specific mutants in the yeast Saccharomyces cerevisiae which per- from disomic (n + 1) strain Z4521-3C. The original disome mitted vegetative replication but blocked the round of used to construct Z4521-3C was provided by Dr. G. Fink (13). replication before meiosis. The mutants synthesized car- Construction and properties of Z4521-3C and details of mu- bohydrate, protein, and RNA during the expected interval tant isolation have been described (12). Z4521-3C and both of premeiotic replication, suggesting that their lesions preferentially affected synthesis of DNA. The mutations mutants have the following general structure: blocked meiosis, as judged by a coincident inhibition of intragenic recombination and ascospore formation. The leu2-27 a lesions were characterized as recessive nuclear genes, and + + + ade2-1, met2, ura3 his 4 leu2- + a (III) were designated mei-1, mei-2, and mei-3; complementa- ade-1,met, ua3his 4 leu 2-1 aa thr 4 tion indicated that the relevant gene products were not p identical.
    [Show full text]
  • HEREDITARY CANCER PANELS Part I
    Pathology and Laboratory Medicine Clinic Building, K6, Core Lab, E-655 2799 W. Grand Blvd. HEREDITARY CANCER PANELS Detroit, MI 48202 855.916.4DNA (4362) Part I- REQUISITION Required Patient Information Ordering Physician Information Name: _________________________________________________ Gender: M F Name: _____________________________________________________________ MRN: _________________________ DOB: _______MM / _______DD / _______YYYY Address: ___________________________________________________________ ICD10 Code(s): _________________/_________________/_________________ City: _______________________________ State: ________ Zip: __________ ICD-10 Codes are required for billing. When ordering tests for which reimbursement will be sought, order only those tests that are medically necessary for the diagnosis and treatment of the patient. Phone: _________________________ Fax: ___________________________ Billing & Collection Information NPI: _____________________________________ Patient Demographic/Billing/Insurance Form is required to be submitted with this form. Most genetic testing requires insurance prior authorization. Due to high insurance deductibles and member policy benefits, patients may elect to self-pay. Call for more information (855.916.4362) Bill Client or Institution Client Name: ______________________________________________________ Client Code/Number: _____________ Bill Insurance Prior authorization or reference number: __________________________________________ Patient Self-Pay Call for pricing and payment options Toll
    [Show full text]
  • DNA Repair with Its Consequences (E.G
    Cell Science at a Glance 515 DNA repair with its consequences (e.g. tolerance and pathways each require a number of apoptosis) as well as direct correction of proteins. By contrast, O-alkylated bases, Oliver Fleck* and Olaf Nielsen* the damage by DNA repair mechanisms, such as O6-methylguanine can be Department of Genetics, Institute of Molecular which may require activation of repaired by the action of a single protein, Biology, University of Copenhagen, Øster checkpoint pathways. There are various O6-methylguanine-DNA Farimagsgade 2A, DK-1353 Copenhagen K, Denmark forms of DNA damage, such as base methyltransferase (MGMT). MGMT *Authors for correspondence (e-mail: modifications, strand breaks, crosslinks removes the alkyl group in a suicide fl[email protected]; [email protected]) and mismatches. There are also reaction by transfer to one of its cysteine numerous DNA repair pathways. Each residues. Photolyases are able to split Journal of Cell Science 117, 515-517 repair pathway is directed to specific Published by The Company of Biologists 2004 covalent bonds of pyrimidine dimers doi:10.1242/jcs.00952 types of damage, and a given type of produced by UV radiation. They bind to damage can be targeted by several a UV lesion in a light-independent Organisms are permanently exposed to pathways. Major DNA repair pathways process, but require light (350-450 nm) endogenous and exogenous agents that are mismatch repair (MMR), nucleotide as an energy source for repair. Another damage DNA. If not repaired, such excision repair (NER), base excision NER-independent pathway that can damage can result in mutations, diseases repair (BER), homologous recombi- remove UV-induced damage, UVER, is and cell death.
    [Show full text]