Managing DNA Polymerases: Coordinating DNA Replication, DNA Repair, and DNA Recombination
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DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli
DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kath, James Evon. 2016. DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ! ! ! ! ! ! ! DNA!polymerase!exchange!and!lesion!bypass!in!Escherichia)coli! ! A!dissertation!presented! by! James!Evon!Kath! to! The!Committee!on!Higher!Degrees!in!Biophysics! ! in!partial!fulfillment!of!the!requirements! for!the!degree!of! Doctor!of!Philosophy! in!the!subject!of! Biophysics! ! Harvard!University! Cambridge,!Massachusetts! October!2015! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ©!2015!L!James!E.!Kath.!Some!Rights!Reserved.! ! This!work!is!licensed!under!the!Creative!Commons!Attribution!3.0!United!States!License.!To! view!a!copy!of!this!license,!visit:!http://creativecommons.org/licenses/By/3.0/us! ! ! Dissertation!Advisor:!Professor!Joseph!J.!Loparo! ! ! !!!!!!!!James!Evon!Kath! ! DNA$polymerase$exchange$and$lesion$bypass$in$Escherichia)coli$ $ Abstract$ ! Translesion! synthesis! (TLS)! alleviates! -
Ubiquitinated Proliferating Cell Nuclear Antigen Activates Translesion DNA Polymerases and REV1
Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases and REV1 Parie Garg and Peter M. Burgers* Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110 Edited by Jerard Hurwitz, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved November 4, 2005 (received for review July 14, 2005) In response to DNA damage, the Rad6͞Rad18 ubiquitin-conjugat- and requiring additional activation by the Cdc7͞Dbf4 protein ing complex monoubiquitinates the replication clamp proliferating kinase that normally functions in cell cycle progression (5, 12). cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of It is this complex pathway that mainly contributes to DNA PCNA is recognized as an essential step in initiating postreplication damage induced mutagenesis in eukaryotic cells. Although repair, the mechanistic relevance of this modification has remained normally involved in lagging strand DNA replication, the high- elusive. Here, we describe a robust in vitro system that ubiquiti- fidelity Pol ␦ is also required for damage-induced mutagenesis. nates yeast PCNA specifically on Lys-164. Significantly, only those The functions of Pol and Rev1 appear to be uniquely confined PCNA clamps that are appropriately loaded around effector DNA to mutagenesis. Pol is an error-prone DNA polymerase that can by its loader, replication factor C, are ubiquitinated. This observa- bypass damage (13). Rev1 is a deoxycytidyl transferase that tion suggests that, in vitro, only PCNA present at stalled replication shows the highest catalytic activity opposite template guanines forks is ubiquitinated. Ubiquitinated PCNA displays the same and abasic sites (14, 15). Rev1 is primarily responsible for replicative functions as unmodified PCNA. -
Jimmunol.0901240.Full.Pdf
A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene Hypermutation This information is current as Keiji Masuda, Rika Ouchida, Yingqian Li, Xiang Gao, of September 24, 2021. Hiromi Mori and Ji-Yang Wang J Immunol published online 8 July 2009 http://www.jimmunol.org/content/early/2009/07/08/jimmuno l.0901240 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2009/07/07/jimmunol.090124 Material 0.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 8, 2009, doi:10.4049/jimmunol.0901240 The Journal of Immunology A Critical Role for REV1 in Regulating the Induction of C:G Transitions and A:T Mutations during Ig Gene Hypermutation Keiji Masuda,* Rika Ouchida,* Yingqian Li,†* Xiang Gao,† Hiromi Mori,* and Ji-Yang Wang1* REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. -
DNA Polymerase V Activity Is Autoregulated by a Novel Intrinsic DNA-Dependent
1 2 DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent 3 ATPase 4 Aysen L. Erdem1, Malgorzata Jaszczur1, Jeffrey G. Bertram1, Roger Woodgate2, Michael M. Cox3 & 5 Myron F. Goodman1 6 1Departments of Biological Sciences and Chemistry, University of Southern California, University 7 Park, Los Angeles, California 90089-2910, USA. 2Laboratory of Genomic Integrity, National 8 Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 9 Maryland 20892-3371, USA. 3Department of Biochemistry, University of Wisconsin-Madison, 10 Madison, Wisconsin 53706, USA. 11 12 Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, 13 is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA 14 synthesis including translesion synthesis (TLS). We have established three features of the roles 15 of ATP and RecA. 1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent 16 ATPase; 2) bound ATP is required for DNA synthesis; 3) pol V Mut function is regulated by 17 ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering 18 dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R 19 mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic 20 property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to 21 single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or 22 autoregulatory mechanism has previously been found for a DNA polymerase. -
Exploring the Non-Canonical Functions of Metabolic Enzymes Peiwei Huangyang1,2 and M
© 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm033365. doi:10.1242/dmm.033365 REVIEW SPECIAL COLLECTION: CANCER METABOLISM Hidden features: exploring the non-canonical functions of metabolic enzymes Peiwei Huangyang1,2 and M. Celeste Simon1,3,* ABSTRACT A key finding from studies of metabolic enzymes is the existence The study of cellular metabolism has been rigorously revisited over the of mechanistic links between their nuclear localization and the past decade, especially in the field of cancer research, revealing new regulation of transcription. By modulating gene expression, insights that expand our understanding of malignancy. Among these metabolic enzymes themselves facilitate adaptation to rapidly insights isthe discovery that various metabolic enzymes have surprising changing environments. Furthermore, they can directly shape a ’ activities outside of their established metabolic roles, including in cell s epigenetic landscape (Kaelin and McKnight, 2013). the regulation of gene expression, DNA damage repair, cell cycle Strikingly, several metabolic enzymes exert completely distinct progression and apoptosis. Many of these newly identified functions are functions in different cellular compartments. Nuclear fructose activated in response to growth factor signaling, nutrient and oxygen bisphosphate aldolase, for example, directly interacts with RNA ́ availability, and external stress. As such, multifaceted enzymes directly polymerase III to control transcription (Ciesla et al., 2014), -
Reca Acts As a Switch to Regulate Polymerase Occupancy in a Moving Replication Fork
RecA acts as a switch to regulate polymerase occupancy in a moving replication fork Chiara Indiania,1, Meghna Patelb, Myron F. Goodmanb, and Mike E. O’Donnellc,1 aManhattan College, Riverdale, NY 10471; bDepartment of Biological Sciences, University of Southern California, Los Angeles, CA 90089; and cHoward Hughes Medical Institute, Rockefeller University, New York, NY 10065 Contributed by Mike O’Donnell, February 19, 2013 (sent for review February 6, 2013) This report discovers a role of Escherichia coli RecA, the cellular regulates DNA polymerase access to the replication fork. Sur- recombinase, in directing the action of several DNA polymerases prisingly, RecA strongly inhibits fork progression by Pol III repli- at the replication fork. Bulk chromosome replication is performed somes, yet RecA stimulates TLS Pol II and Pol IV replisomes. The by DNA polymerase (Pol) III. However, E. coli contains translesion mechanism that underlies the opposite effects of RecA on Pol III synthesis (TLS) Pols II, IV, and V that also function with the helicase, and the TLS Pol replisomes involves single-strand binding protein primase, and sliding clamp in the replisome. Surprisingly, we find (SSB). Although all of the polymerases function with SSB on the that RecA specifically activates replisomes that contain TLS Pols. In template strand, SSB inhibits TLS Pol function in the context of sharp contrast, RecA severely inhibits the Pol III replisome. Given a replisome, presumably acting in trans by binding lagging-strand the opposite effects of RecA on Pol III and TLS replisomes, we pro- ssDNA. RecA relieves the SSB induced repression of TLS Pol pose that RecA acts as a switch to regulate the occupancy of poly- replisomes and stimulates their action, while inhibiting the Pol III merases within a moving replisome. -
Anti-MAD2L2 Monoclonal Antibody, Clone FQS24768 (DCABH-6886) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
Anti-MAD2L2 monoclonal antibody, clone FQS24768 (DCABH-6886) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Product Overview Rabbit Monoclonal to Mad2L2 Antigen Description Adapter protein able to interact with different proteins and involved in different biological processes. Mediates the interaction between the error-prone DNA polymerase zeta catalytic subunit REV3L and the inserter polymerase REV1, thereby mediating the second polymerase switching in translesion DNA synthesis. Translesion DNA synthesis releases the replication blockade of replicative polymerases, stalled in presence of DNA lesions. May also regulate another aspect of cellular response to DNA damage through regulation of the JNK-mediated phosphorylation and activation of the transcriptional activator ELK1. Inhibits the FZR1- and probably CDC20-mediated activation of the anaphase promoting complex APC thereby regulating progression through the cell cycle. Regulates TCF7L2-mediated gene transcription and may play a role in epithelial-mesenchymal transdifferentiation. Immunogen Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Mad2L2 aa 150 to the C-terminus. The exact sequence is proprietary.Database link: Q9UI95 Isotype IgG Source/Host Rabbit Species Reactivity Mouse, Rat, Human Clone FQS24768 Purity Protein A purified Conjugate Unconjugated Applications IP, ICC/IF, Flow Cyt, IHC-P, WB Positive Control K562, SW480, Hela, Molt-4 cell lysates, human ovarian carcinoma tissue, Hela cells, Format Liquid Size 100 μl Buffer Preservative: 0.01% Sodium azide; Constituents: 59% PBS, 0.05% BSA, 40% Glycerol 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved Preservative 0.01% Sodium Azide Storage Store at +4°C short term (1-2 weeks). -
Arthur Kornberg Discovered (The First) DNA Polymerase Four
Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E. -
DNA Polymerase Ι Compensates for Fanconi Anemia Pathway Deficiency by Countering DNA Replication Stress
DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress Rui Wanga, Walter F. Lenoirb,c, Chao Wanga, Dan Sua, Megan McLaughlinb, Qianghua Hua, Xi Shena, Yanyan Tiana, Naeh Klages-Mundta,c, Erica Lynna, Richard D. Woodc,d, Junjie Chena,c, Traver Hartb,c, and Lei Lia,c,e,1 aDepartment of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; bDepartment of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; cThe University of Texas MD Anderson Cancer Center University of Texas Health Science Center at Houston Graduate School of Biomedical Sciences, Houston, TX 77030; dDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; and eLife Sciences Institute, Zhejiang University, Hangzhou, China 310058 Edited by Wei Yang, NIH, Bethesda, MD, and approved November 12, 2020 (received for review May 8, 2020) Fanconi anemia (FA) is caused by defects in cellular responses to proteins, required in homologous recombination (FANCD1/ DNA crosslinking damage and replication stress. Given the con- BRCA2, FANCO/RAD51C, FANCJ/BARD1, and FANCR/ stant occurrence of endogenous DNA damage and replication fork RAD51) (22–26). stress, it is unclear why complete deletion of FA genes does not In addition to the direct role in crosslinking damage repair, have a major impact on cell proliferation and germ-line FA patients FA pathway components are linked to the protection of repli- are able to progress through development well into their adult- cation fork integrity during replication interruption that is not hood. -
Y-Family DNA Polymerases in Escherichia Coli
Y-family DNA polymerases in Escherichia coli The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Jarosz, Daniel F. et al. “Y-family DNA Polymerases in Escherichia Coli.” Trends in Microbiology 15.2 (2007): 70–77. Web. 13 Apr. 2012. © 2007 Elsevier Ltd. As Published http://dx.doi.org/10.1016/j.tim.2006.12.004 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/70041 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Review TRENDS in Microbiology Vol.15 No.2 Y-family DNA polymerases in Escherichia coli Daniel F. Jarosz1, Penny J. Beuning2,3, Susan E. Cohen2 and Graham C. Walker2 1 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3 Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA The observation that mutations in the Escherichia coli that is also lexA-independent [4]. This might have genes umuC+ and umuD+ abolish mutagenesis induced particularly important implications for bacteria living by UV light strongly supported the counterintuitive under conditions of nutrient starvation. The SOS response notion that such mutagenesis is an active rather than also seems to be oscillatory at the single-cell level, and this passive process. Genetic and biochemical studies have oscillation is dependent on the umuDC genes [5]. -
DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of Dntp Pools
University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of dNTP Pools Olga V. Kochenova University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biochemistry Commons, Genetics Commons, Molecular Biology Commons, and the Molecular Genetics Commons Recommended Citation Kochenova, Olga V., "DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error- Prone Synthesis, and the Role of dNTP Pools" (2016). Theses & Dissertations. 153. https://digitalcommons.unmc.edu/etd/153 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. DNA POLYMERASE ζ -DEPENDENT MUTAGENESIS: MOLECULAR SPECIFICITY, EXTENT OF ERROR-PRONE SYNTHESIS, AND THE ROLE OF dNTP POOLS by Olga Kochenova A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Cancer Research Graduate Program Under the Supervision of Professor Polina V. Shcherbakova University of Nebraska Medical Center Omaha, Nebraska October, 2016 Supervisory Committee: Youri Pavlov, Ph. D. Tadayoshi Bessho, Ph. D. Michel Ouellette, Ph. D. DNA POLYMERASE ζ -DEPENDENT MUTAGENESIS: MOLECULAR SPECIFICITY, EXTENT OF ERROR-PRONE SYNTHESIS, AND THE ROLE OF dNTP POOLS Olga V. Kochenova, Ph.D. University of Nebraska, 2016 Supervisor: Polina V. Shcherbakova, Ph.D. Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. -
Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass
cancers Review Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass Nicole A. Wilkinson y, Katherine S. Mnuskin y, Nicholas W. Ashton * and Roger Woodgate * Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, USA; [email protected] (N.A.W.); [email protected] (K.S.M.) * Correspondence: [email protected] (N.W.A.); [email protected] (R.W.); Tel.: +1-301-435-1115 (N.W.A.); +1-301-435-0740 (R.W.) Co-first authors. y Received: 29 August 2020; Accepted: 29 September 2020; Published: 2 October 2020 Simple Summary: Ubiquitin and ubiquitin-like proteins are conjugated to many other proteins within the cell, to regulate their stability, localization, and activity. These modifications are essential for normal cellular function and the disruption of these processes contributes to numerous cancer types. In this review, we discuss how ubiquitin and ubiquitin-like proteins regulate the specialized replication pathways of DNA damage bypass, as well as how the disruption of these processes can contribute to cancer development. We also discuss how cancer cell survival relies on DNA damage bypass, and how targeting the regulation of these pathways by ubiquitin and ubiquitin-like proteins might be an effective strategy in anti-cancer therapies. Abstract: Many endogenous and exogenous factors can induce genomic instability in human cells, in the form of DNA damage and mutations, that predispose them to cancer development. Normal cells rely on DNA damage bypass pathways such as translesion synthesis (TLS) and template switching (TS) to replicate past lesions that might otherwise result in prolonged replication stress and lethal double-strand breaks (DSBs).