scholarly journals MutS regulates access of the error-prone DNA polymerase Pol IV to replication sites: a novel mechanism for maintaining replication fidelity

2016 ◽  
Vol 44 (16) ◽  
pp. 7700-7713 ◽  
Author(s):  
Lucía M. Margara ◽  
Marisa M. Fernández ◽  
Emilio L. Malchiodi ◽  
Carlos E. Argaraña ◽  
Mariela R. Monti
Keyword(s):  
Dna Polymerase ◽  
Replication Fidelity ◽  
Pol Iv ◽  
Replication Sites

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals The Roles of Tyr391 and Tyr619 in RB69 DNA Polymerase Replication Fidelity

2007 ◽  
Vol 368 (1) ◽  
pp. 18-29 ◽  
Author(s):  
Agata Jacewicz ◽  
Karolina Makiela ◽  
Andrzej Kierzek ◽  
John W. Drake ◽  
Anna Bebenek
Keyword(s):  
Dna Polymerase ◽  
Replication Fidelity

scholarly journals Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase   replication fidelity

2008 ◽  
Vol 36 (7) ◽  
pp. 2174-2181 ◽  
Author(s):  
Z. F. Pursell ◽  
J. T. McDonald ◽  
C. K. Mathews ◽  
T. A. Kunkel
Keyword(s):  
Dna Polymerase ◽  
Replication Fidelity

scholarly journals The Enhanced DNA Replication Fidelity of a Mutant Herpes Simplex Virus Type 1 DNA Polymerase Is Mediated by an Improved Nucleotide Selectivity and Reduced Mismatch Extension Ability

2008 ◽  
Vol 82 (17) ◽  
pp. 8937-8941 ◽  
Author(s):  
Wang Tian ◽  
Ying T. Hwang ◽  
Charles B. C. Hwang
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Replication Fidelity ◽  
Simplex Virus ◽  
Dna Replication Fidelity

ABSTRACT We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.

scholarly journals Escherichia coli DNA Polymerase IV Mutator Activity: Genetic Requirements and Mutational Specificity

2000 ◽  
Vol 182 (16) ◽  
pp. 4587-4595 ◽  
Author(s):  
Jérôme Wagner ◽  
Takehiko Nohmi
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Amino Acid Level ◽  
Nucleotide Substitutions ◽  
Pol Iv ◽  
Mutator Activity ◽  
Sequence Homologies ◽  
In The Wild ◽  
Dna Polymerase Iv ◽  
Dna Pol Iv

ABSTRACT The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of λ phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F′ plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase η) ofSaccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of λ phage. The results indicated that the DNA Pol IV mutator activity is independent ofpolA, polB, recA,umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5′-GX-3′ sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.

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