scholarly journals Escherichia coli DNA Polymerase IV (Pol IV), but Not Pol II, Dynamically Switches with a Stalled Pol III* Replicase

2012 ◽  
Vol 194 (14) ◽  
pp. 3589-3600 ◽  
Author(s):  
J. M. H. Heltzel ◽  
R. W. Maul ◽  
D. W. Wolff ◽  
M. D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Pol Ii ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Pol Iii

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 Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells.

2005 ◽  
Vol 52 (1) ◽  
pp. 139-147
Author(s):  
Michał Wrzesiński ◽  
Anetta Nowosielska ◽  
Jadwiga Nieminuszczy ◽  
Elzbieta Grzesiuk
Keyword(s):  
Escherichia Coli ◽  
Uv Light ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Uv Mutagenesis ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Induced Mutagenesis ◽  
Pol Iii

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

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.

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 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 Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli: Analysis of the dnaX36 Mutator Mutant

2007 ◽  
Vol 190 (5) ◽  
pp. 1730-1742 ◽  
Author(s):  
Damian Gawel ◽  
Phuong T. Pham ◽  
Iwona J. Fijalkowska ◽  
Piotr Jonczyk ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Gene Dosage ◽  
Dna Polymerases ◽  
Dependent Manner ◽  
Mutator Phenotype ◽  
Pol Ii ◽  
Pol Iv ◽  
High Fraction ◽  
Pol Iii

ABSTRACT The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication.

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.

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.

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