scholarly journals Role of Escherichia coli DNA Polymerase IV in In Vivo Replication Fidelity

2004 ◽  
Vol 186 (14) ◽  
pp. 4802-4807 ◽  
Author(s):  
Wojciech Kuban ◽  
Piotr Jonczyk ◽  
Damian Gawel ◽  
Karolina Malanowska ◽  
Roel M. Schaaper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Error Rate ◽  
The Other ◽  
Chromosomal Dna ◽  
Pol Iv ◽  
Dna Polymerase Iv

ABSTRACT We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F′(pro-lac) episome.

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 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.

Role of the 5´ → 3´ exonuclease and Klenow fragment of Escherichia coli DNA polymerase I in base mismatch repair

2007 ◽  
Vol 278 (2) ◽  
pp. 211-220 ◽  
Author(s):  
Masaru Imai ◽  
Yu-ichiro Tago ◽  
Makoto Ihara ◽  
Masakado Kawata ◽  
Kazuo Yamamoto
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
Base Mismatch ◽  
Polymerase I

scholarly journals Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in Escherichia coli

2003 ◽  
Vol 185 (11) ◽  
pp. 3469-3472 ◽  
Author(s):  
Joshua D. Tompkins ◽  
Jennifer L. Nelson ◽  
Jill C. Hazel ◽  
Stacy L. Leugers ◽  
Jeffrey D. Stumpf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Adaptive Mutation ◽  
Wild Type ◽  
Pol Iv ◽  
In The Wild ◽  
Dna Polymerase Iv

ABSTRACT The frequencies of nonselected mutations among adaptive Lac+ revertants of Escherichia coli strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.

scholarly journals Roles of the Escherichia coli RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In Vivo

2005 ◽  
Vol 187 (22) ◽  
pp. 7607-7618 ◽  
Author(s):  
Robert W. Maul ◽  
Mark D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Reca Protein ◽  
Sos Response ◽  
Mutant Form ◽  
Elevated Expression ◽  
Dna Polymerase Iv ◽  
Dna Polymerase Ii

ABSTRACT The Escherichia coli β sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the dnaN159 allele, which encodes a mutant form of the β clamp (β159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the five E.coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the dinB-encoded polymerase IV is sufficient to result in conditional lethality of the dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., ΔrecR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., ΔpolB or ΔumuDC). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.

scholarly journals Separate DNA Pol II- and Pol IV-Dependent Pathways of Stress-Induced Mutation during Double-Strand-Break Repair in Escherichia coli Are Controlled by RpoS

2010 ◽  
Vol 192 (18) ◽  
pp. 4694-4700 ◽  
Author(s):  
Ryan L. Frisch ◽  
Yang Su ◽  
P. C. Thornton ◽  
Janet L. Gibson ◽  
Susan M. Rosenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Induced Mutations ◽  
Pol Ii ◽  
Pol Iv ◽  
Break Repair ◽  
Dna Polymerase Iv

ABSTRACT Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.

scholarly journals Role of DNA Polymerase IV in Escherichia coli SOS Mutator Activity

2006 ◽  
Vol 188 (22) ◽  
pp. 7977-7980 ◽  
Author(s):  
Wojciech Kuban ◽  
Magdalena Banach-Orlowska ◽  
Roel M. Schaaper ◽  
Piotr Jonczyk ◽  
Iwona J. Fijalkowska
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Dna Polymerase ◽  
Constitutive Expression ◽  
Significant Fraction ◽  
Mutator Phenotype ◽  
Mutator Activity ◽  
Dna Polymerase V ◽  
Dna Polymerase Iv

ABSTRACT Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).

scholarly journals Mismatch repair and DNA polymerase δ proofreading prevent catastrophic accumulation of leading strand errors in cells expressing a cancer-associated DNA polymerase ϵ variant

2020 ◽  
Vol 48 (16) ◽  
pp. 9124-9134 ◽  
Author(s):  
Chelsea R Bulock ◽  
Xuanxuan Xing ◽  
Polina V Shcherbakova
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Specific Manner ◽  
Leading Strand ◽  
The Impact ◽  
Increased Activity

Abstract Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo−) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo− at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδ proofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδ proofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδ exonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.

scholarly journals RpoS, the Stress Response Sigma Factor, Plays a Dual Role in the Regulation of Escherichia coli's Error-Prone DNA Polymerase IV

2010 ◽  
Vol 192 (14) ◽  
pp. 3639-3644 ◽  
Author(s):  
Kimberly A. M. Storvik ◽  
Patricia L. Foster
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Stationary Phase ◽  
Dna Polymerase ◽  
Sigma Factor ◽  
Dna Lesions ◽  
Indirect Pathway ◽  
General Stress Response ◽  
Pol Iv ◽  
Dna Polymerase Iv

ABSTRACT RpoS, Escherichia coli's general stress response sigma factor, regulates error-prone DNA polymerase IV (Pol IV) (encoded by the dinB gene). Pol IV is induced in stationary-phase cells, and thereafter, levels of the protein remain elevated for several days of continuous incubation. This induction and persistence in stationary-phase cells are dependent on RpoS. Data presented here show that this regulation is direct via the RpoS-directed transcription of the dinB gene. However, a loss of RpoS also results in a decrease in Pol IV-dependent mutation when Pol IV is overexpressed from an RpoS-independent promoter in exponentially growing cells. The loss of RpoS also increases cell sensitivity to 4-nitroquinoline-1-oxide, indicating that RpoS affects the ability of Pol IV to bypass DNA lesions. Thus, in addition to directly driving the transcription of the dinB gene in stationary-phase cells, RpoS regulates the activity of Pol IV in exponentially growing cells via a second, indirect pathway.

Sign in / Sign up

Export Citation Format

Share Document