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.

DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct

2001 ◽  
Vol 29 (2) ◽  
pp. 191-195 ◽  
Author(s):  
R. P. P. Fuchs ◽  
N. Koffel-Schwartz ◽  
S. Pelet ◽  
R. Janel-Bintz ◽  
R. Napolitano ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Cellular Response ◽  
Uv Light ◽  
Hot Spot ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Pol Ii ◽  
Frameshift Mutations ◽  
Replication Intermediate ◽  
Pol V

The Nar I sequence represents a strong mutation hot spot for - 2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5′-GGCGCC → GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced - 2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.

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 Steric Gate Variants of UmuC Confer UV Hypersensitivity on Escherichia coli

2008 ◽  
Vol 191 (15) ◽  
pp. 4815-4823 ◽  
Author(s):  
Brenna W. Shurtleff ◽  
Jaylene N. Ollivierre ◽  
Mohammad Tehrani ◽  
Graham C. Walker ◽  
Penny J. Beuning
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Uv Light ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Dominant Negative ◽  
Gene Encoding ◽  
Futile Cycle ◽  
Y Family ◽  
Negative Phenotype

ABSTRACT Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the β processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

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 The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 817-831 ◽  
Author(s):  
R H Schiestl ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Gamma Ray ◽  
Dna Lesions ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Uv Mutagenesis ◽  
Uv Sensitivity ◽  
Suppressor Mutations ◽  
Induced Mutagenesis

Abstract rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

scholarly journals Escherichia coli DNA Polymerase III Can Replicate Efficiently past a T-T cis-syn Cyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded by dnaQ Are Inactivated

2002 ◽  
Vol 184 (10) ◽  
pp. 2674-2681 ◽  
Author(s):  
Angela Borden ◽  
Paul I. O'Grady ◽  
Dominique Vandewiele ◽  
Antonio R. Fernández de Henestrosa ◽  
Christopher W. Lawrence ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Wild Type ◽  
Lesion Bypass ◽  
Base Pairs ◽  
Pol V ◽  
Pol I ◽  
Cyclobutane Dimer ◽  
Dna Polymerase V ◽  
Pol Iii

ABSTRACT Although very little replication past a T-T cis-syn cyclobutane dimer normally takes place in Escherichia coli in the absence of DNA polymerase V (Pol V), we previously observed as much as half of the wild-type bypass frequency in Pol V-deficient (ΔumuDC) strains if the 3′ to 5′ exonuclease proofreading activity of the Pol III ε subunit was also disabled by mutD5. This observation might be explained in at least two ways. In the absence of Pol V, wild-type Pol III might bind preferentially to the blocked primer terminus but be incapable of bypass, whereas the proofreading-deficient enzyme might dissociate more readily, providing access to bypass polymerases. Alternatively, even though wild-type Pol III is generally regarded as being incapable of lesion bypass, proofreading-impaired Pol III might itself perform this function. We have investigated this issue by examining dimer bypass frequencies in ΔumuDC mutD5 strains that were also deficient for Pol I, Pol II, and Pol IV, both singly and in all combinations. Dimer bypass frequencies were not decreased in any of these strains and indeed in some were increased to levels approaching those found in strains containing Pol V. Efficient dimer bypass was, however, entirely dependent on the proofreading deficiency imparted by mutD5, indicating the surprising conclusion that bypass was probably performed by the mutD5 Pol III enzyme itself. This mutant polymerase does not replicate past the much more distorted T-T (6-4) photoadduct, however, suggesting that it may only replicate past lesions, like the T-T dimer, that form base pairs normally.

scholarly journals RNA polymerases IV and V influence the 3’ boundaries of Polymerase II transcription units in Arabidopsis

2017 ◽  
Author(s):  
Anastasia McKinlay ◽  
Ram Podicheti ◽  
Jered M. Wendte ◽  
Ross Cocklin ◽  
Douglas B. Rusch
Keyword(s):  
Cytosine Methylation ◽  
De Novo ◽  
Transcription Termination ◽  
Rna Polymerases ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol Iv ◽  
Pol V ◽  
Genomic Repeats

AbstractNuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3’ boundaries of Pol II transcription units. Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II – transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination by influencing polymerase bypass or release at collision sites for convergent genes.

scholarly journals Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination

2017 ◽  
Author(s):  
Michelle Marasco ◽  
Weiyi Li ◽  
Michael Lynch ◽  
Craig S. Pikaard
Keyword(s):  
Rna Synthesis ◽  
Catalytic Properties ◽  
Rna Polymerases ◽  
Catalytic Subunits ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Nucleotide Incorporation ◽  
In Trans ◽  
Conserved Amino Acids

AbstractCatalytic subunits of DNA-dependent RNA polymerases of bacteria, archaea and eukaryotes share hundreds of ultra-conserved amino acids. Remarkably, the plant-specific RNA silencing enzymes, Pol IV and Pol V differ from Pols I, II and III at ~140 of these positions, yet remain capable of RNA synthesis. Whether these amino acid changes in Pols IV and V alter their catalytic properties in comparison to Pol II, from which they evolved, is unknown. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is notably error-prone, which may be tolerable, or even beneficial, for biosynthesis of siRNAs targeting transposon families in trans. By contrast, Pol V exhibits high fidelity transcription, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci in order to recruit siRNA-Argonaute protein silencing complexes.

AlkB dioxygenase in preventing MMS-induced mutagenesis in Escherichia coli: Effect of Pol V and AlkA proteins

DNA Repair ◽  
2006 ◽  
Vol 5 (2) ◽  
pp. 181-188 ◽  
Author(s):  
Jadwiga Nieminuszczy ◽  
Anna Sikora ◽  
Michał Wrzesiński ◽  
Celina Janion ◽  
Elżbieta Grzesiuk
Keyword(s):  
Escherichia Coli ◽  
Pol V ◽  
Induced Mutagenesis
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