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 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 (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 Polymerases Leave Fingerprints: Analysis of the Mutational Spectrum in Escherichia coli rpoB To Assess the Role of Polymerase IV in Spontaneous Mutation

2004 ◽  
Vol 186 (9) ◽  
pp. 2900-2905 ◽  
Author(s):  
Erika Wolff ◽  
Mandy Kim ◽  
Kaibin Hu ◽  
Hanjing Yang ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Exponential Growth ◽  
Hot Spots ◽  
Liquid Culture ◽  
Spontaneous Mutation ◽  
Mutational Spectrum ◽  
Pol Iv ◽  
Rifampin Resistance ◽  
Plasmid Amplification

ABSTRACT We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F′128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.

Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2487-2497 ◽  
Author(s):  
Anne Vianney ◽  
Grégory Jubelin ◽  
Sophie Renault ◽  
Corine Dorel ◽  
Philippe Lejeune ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Membrane Integrity ◽  
Regulatory Proteins ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Dominant Effect ◽  
Gram Negative ◽  
E Coli

Curli are necessary for the adherence of Escherichia coli to surfaces, and to each other, during biofilm formation, and the csgBA and csgDEFG operons are both required for their synthesis. A recent survey of gene expression in Pseudomonas aeruginosa biofilms has identified tolA as a gene activated in biofilms. The tol genes play a fundamental role in maintaining the outer-membrane integrity of Gram-negative bacteria. RcsC, the sensor of the RcsBCD phosphorelay, is involved, together with RcsA, in colanic acid capsule synthesis, and also modulates the expression of tolQRA and csgDEFG. In addition, the RcsBCD phosphorelay is activated in tol mutants or when Tol proteins are overexpressed. These results led the authors to investigate the role of the tol genes in biofilm formation in laboratory and clinical isolates of E. coli. It was shown that the adherence of cells was lowered in the tol mutants. This could be the result of a drastic decrease in the expression of the csgBA operon, even though the expression of csgDEFG was slightly increased under such conditions. It was also shown that the Rcs system negatively controls the expression of the two csg operons in an RcsA-dependent manner. In the tol mutants, activation of csgDEFG occurred via OmpR and was dominant upon repression by RcsB and RcsA, while these two regulatory proteins repressed csgBA through a dominant effect on the activator protein CsgD, thus affecting curli synthesis. The results demonstrate that the Rcs system, previously known to control the synthesis of the capsule and the flagella, is an additional component involved in the regulation of curli. Furthermore, it is shown that the defect in cell motility observed in the tol mutants depends on RcsB and RcsA.

scholarly journals Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs

1998 ◽  
Vol 180 (21) ◽  
pp. 5712-5717 ◽  
Author(s):  
Malgorzata M. Slupska ◽  
Angela G. King ◽  
Louise I. Lu ◽  
Rose H. Lin ◽  
Emily F. Mao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aspartic Acid ◽  
Dna Polymerase ◽  
Mutation Rate ◽  
Mutator Phenotype ◽  
Polymerase Iii ◽  
In Series ◽  
Mutator Effect

ABSTRACT We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, ɛ, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered ɛ subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutDalleles. In the present work, we extended the study of specificmutD alleles and constructed 16 altered mutDgenes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes ɛ. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the ɛ subunit playing a major role in the mutator effect of misreading tRNAs.

scholarly journals Modulation of Yeast Genome Expression in Response to Defective RNA Polymerase III-Dependent Transcription

2005 ◽  
Vol 25 (19) ◽  
pp. 8631-8642 ◽  
Author(s):  
Christine Conesa ◽  
Roberta Ruotolo ◽  
Pascal Soularue ◽  
Tiffany A. Simms ◽  
David Donze ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Dosage ◽  
Rna Polymerase Iii ◽  
Yeast Cells ◽  
Yeast Genome ◽  
Altered Expression ◽  
Pol Ii ◽  
Initiator Trna ◽  
Genome Expression ◽  
Pol Iii

ABSTRACT We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNAMet. Initiator tRNAMet depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes.

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 Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

2010 ◽  
Vol 192 (8) ◽  
pp. 2220-2227 ◽  
Author(s):  
Bavesh D. Kana ◽  
Garth L. Abrahams ◽  
Nackmoon Sung ◽  
Digby F. Warner ◽  
Bhavna G. Gordhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Alkylating Agents ◽  
Spontaneous Mutation ◽  
Dna Polymerases ◽  
Binding Motif ◽  
Dependent Manner ◽  
Mouse Macrophages ◽  
Y Family

ABSTRACT The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N 2-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

scholarly journals Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Indira T. Kudva ◽  
Michelle Q. Carter ◽  
Vijay K. Sharma ◽  
Judith A. Stasko ◽  
Jorge A. Giron
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Human Cell ◽  
Control Strategies ◽  
Dependent Manner ◽  
Human Cell Lines ◽  
O157 Strain ◽  
Content Type

ABSTRACT Our recent studies have shown that intimin and the locus of enterocyte effacement-encoded proteins do not play a role in Escherichia coli O157:H7 (O157) adherence to the bovine recto-anal junction squamous epithelial (RSE) cells. To define factors that play a contributory role, we investigated the role of curli, fimbrial adhesins commonly implicated in adherence to various fomites and plant and human epithelial cells, in O157 adherence to RSE cells. Specifically, we examined (i) wild-type strains of O157; (ii) curli variants of O157 strains; (iii) isogenic curli deletion mutants of O157; and (iv) adherence inhibition of O157 using anti-curlin sera. Results of these experiments conducted under stringent conditions suggest that curli do not solely contribute to O157 adherence to RSE cells and in fact demonstrate a modulating effect on O157 adherence to RSE cells in contrast to HEp-2 cells (human epidermoid carcinoma of the larynx cells with HeLa contamination). The absence of curli and presence of blocking anti-curli antibodies enhanced O157-RSE cell interactions among some strains, thus alluding to a spatial, tempering effect of curli on O157 adherence to RSE cells when present. At the same time, the presence or absence of curli did not alter RSE cell adherence patterns of another O157 strain. These observations are at variance with the reported role of curli in O157 adherence to human cell lines such as HEp-2 and need to be factored in when developing anti-adherence modalities for preharvest control of O157 in cattle. IMPORTANCE This study demonstrated that O157 strains interact with epithelial cells in a host-specific manner. The fimbriae/adhesins that are significant for adherence to human cell lines may not have a role or may have a modulating role in O157 adherence to bovine cells. Targeting such adhesins may not prevent O157 attachment to bovine cells but instead may result in improved adherence. Hence, conducting host-specific evaluations is critical when selecting targets for O157 control strategies.

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