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1.
Nucleic Acids Res ; 51(4): 1766-1782, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36762476

RESUMO

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase-DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand.


Assuntos
Reparo do DNA , Escherichia coli , DNA Polimerase III/genética , Replicação do DNA , Escherichia coli/genética , Ribonucleotídeos/metabolismo
2.
PLoS Genet ; 15(12): e1008494, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815930

RESUMO

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction.


Assuntos
Instabilidade Genômica , Sequências Repetitivas de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
PLoS Genet ; 13(1): e1006572, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28107343

RESUMO

To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants.


Assuntos
DNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA Polimerase II/genética , Mutação , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
4.
Int J Mol Sci ; 21(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322195

RESUMO

The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.


Assuntos
DNA Helicases/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Proteínas Nucleares/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , DNA Polimerase II/genética , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutagênese , Mutação , Taxa de Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Mutações Sintéticas Letais/genética
5.
Mol Microbiol ; 92(4): 659-80, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24628792

RESUMO

The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Complexos Multienzimáticos/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética
6.
Mutat Res ; 759: 22-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24269257

RESUMO

The fidelity with which organisms replicate their chromosomal DNA is of considerable interest. Detailed studies in the bacterium Escherichia coli have indicated that the fidelity of leading- and lagging-strand DNA replication is not the same, based on experiments in which the orientation of certain mutational targets on the chromosome was inverted relative to the movement of the replication fork: different mutation rates for several base-pair substitutions were observed depending on this orientation. While these experiments are indicative of differential replication fidelity in the two strands, a conclusion whether leading or lagging strand is the more accurate depends on knowledge of the primary mispairing error responsible for the base substitutions in question. A broad analysis of in vitro base-pairing preferences of DNA polymerases led us to propose that lagging-strand is the more accurate strand. In the present work, we present more direct in vivo evidence in support of this proposal. We determine the orientation dependence of mutant frequencies in ndk and dcd strains, which carry defined dNTP pool alterations. As these pool alterations lead to predictable effects on the array of possible mispairing errors, they mark the strands in which the observed errors occur. The combined results support the proposed higher accuracy of lagging-strand replication in E. coli.


Assuntos
Replicação do DNA , Desoxirribonucleotídeos/metabolismo , Escherichia coli/genética , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Óperon Lac , Nucleotídeos de Timina/metabolismo
7.
Mutat Res ; 737(1-2): 34-42, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22709919

RESUMO

The Saccharomyces cerevisiae DNA polymerase epsilon holoenzyme (Pol ɛ HE) is composed of four subunits: Pol2p, Dpb2p, Dpb3p and Dpb4p. The biological functions of Pol2p, the catalytic subunit of Pol ɛ, are subject of active investigation, while the role of the other three, noncatalytic subunits, is not well defined. We showed previously that mutations in Dpb2p, a noncatalytic but essential subunit of Pol ɛ HE, influence the fidelity of DNA replication in yeast cells. The strength of the mutator phenotype due to the different dpb2 alleles was inversely proportional to the strength of protein-protein interactions between Pol2p and the mutated forms of Dpb2p. To understand better the mechanisms of the contribution of Dpb2p to the controlling of the level of spontaneous mutagenesis we undertook here a further genetic analysis of the mutator phenotype observed in dpb2 mutants. We demonstrate that the presence of mutated forms of Dpb2p in the cell not only influences the intrinsic fidelity of Pol ɛ but also facilitates more frequent participation of error-prone DNA polymerase zeta (Pol ζ) in DNA replication. The obtained results suggest that the structural integrity of Pol ɛ HE is a crucial contributor to accurate chromosomal DNA replication and, when compromised, favors participation of error prone DNA Pol ζ in this process.


Assuntos
DNA Polimerase II/química , Replicação do DNA , Mutagênese , Proteínas de Saccharomyces cerevisiae/fisiologia , DNA Polimerase II/fisiologia , DNA Fúngico/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Saccharomyces cerevisiae/genética
8.
J Bacteriol ; 193(1): 296-300, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21036999

RESUMO

The Escherichia coli dnaX36 mutant displays a mutator effect, reflecting a fidelity function of the dnaX-encoded τ subunit of the DNA polymerase III (Pol III) holoenzyme. We have shown that this fidelity function (i) applies to both leading- and lagging-strand synthesis, (ii) is independent of Pol IV, and (iii) is limited by Pol II.


Assuntos
DNA Polimerase III/metabolismo , Replicação do DNA/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fatores de Transcrição/metabolismo , Cromossomos Bacterianos , DNA Polimerase III/genética , Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutação , Fatores de Transcrição/genética
9.
Mol Microbiol ; 74(5): 1114-27, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19843230

RESUMO

We have investigated the possible role of Escherichia coli DNA polymerase (Pol) I in chromosomal replication fidelity. This was done by substituting the chromosomal polA gene by the polAexo variant containing an inactivated 3'-->5' exonuclease, which serves as a proofreader for this enzyme's misinsertion errors. Using this strain, activities of Pol I during DNA replication might be detectable as increases in the bacterial mutation rate. Using a series of defined lacZ reversion alleles in two orientations on the chromosome as markers for mutagenesis, 1.5- to 4-fold increases in mutant frequencies were observed. In general, these increases were largest for lac orientations favouring events during lagging strand DNA replication. Further analysis of these effects in strains affected in other E. coli DNA replication functions indicated that this polAexo mutator effect is best explained by an effect that is additive compared with other error-producing events at the replication fork. No evidence was found that Pol I participates in the polymerase switching between Pol II, III and IV at the fork. Instead, our data suggest that the additional errors produced by polAexo are created during the maturation of Okazaki fragments in the lagging strand.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Escherichia coli/enzimologia , DNA , DNA Polimerase I/genética , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Exodesoxirribonucleases/metabolismo , Dados de Sequência Molecular
10.
Genetics ; 178(2): 633-47, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18245343

RESUMO

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.


Assuntos
DNA Polimerase II/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Polimerase II/metabolismo , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Genótipo , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Mutat Res ; 669(1-2): 27-35, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19463834

RESUMO

Most of the prokaryotic and eukaryotic replicative polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells.


Assuntos
DNA Polimerase II/metabolismo , Mutação/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sobrevivência Celular , Deleção de Genes , Immunoblotting , Mutagênese , Fenótipo , Subunidades Proteicas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-Híbrido
12.
DNA Repair (Amst) ; 83: 102643, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31324532

RESUMO

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.


Assuntos
Alelos , DNA Polimerase III/genética , Replicação do DNA , Escherichia coli/genética , Mutação , DNA Polimerase beta/metabolismo , Fenótipo
13.
DNA Repair (Amst) ; 84: 102685, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31543434

RESUMO

pol VICE391 (RumA'2B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous "SOS-induced" mutagenesis than the related E. coli pol V (UmuD'2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC. We have generated a steric gate pol VICE391 variant (pol VICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol VICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome. Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol VICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Instabilidade Genômica , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutação , Domínios Proteicos , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo
14.
J Bacteriol ; 190(5): 1730-42, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18156258

RESUMO

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 tau 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 tau, essential for interaction of tau with the alpha (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered alpha-tau 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 tau subunit, in securing a high fidelity of replication.


Assuntos
Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Mutação , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/genética , DNA-Formamidopirimidina Glicosilase/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Fenótipo , Ligação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
DNA Repair (Amst) ; 29: 23-35, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25758782

RESUMO

DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ's biochemical properties including catalytic efficiency, processivity or proofreading activity - it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase II/genética , Replicação do DNA/genética , Mutação , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Polimerase II/metabolismo , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
Mutat Res ; 501(1-2): 129-36, 2002 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-11934444

RESUMO

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.


Assuntos
Replicação do DNA/genética , Escherichia coli/genética , Mutação da Fase de Leitura/genética , Óperon Lac/genética , Mutagênese
17.
FEMS Microbiol Rev ; 36(6): 1105-21, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22404288

RESUMO

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.


Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA Bacteriano/genética
18.
J Bacteriol ; 188(22): 7977-80, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16980447

RESUMO

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).


Assuntos
DNA Polimerase beta/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese , Resposta SOS em Genética
19.
Mol Microbiol ; 58(1): 61-70, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16164549

RESUMO

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.


Assuntos
DNA Polimerase II/fisiologia , Replicação do DNA/fisiologia , DNA Bacteriano/biossíntese , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Alelos , Ligação Competitiva , Cromossomos Bacterianos , DNA Polimerase III/genética , DNA Polimerase III/fisiologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos , Genes Reporter , Mutação , beta-Galactosidase/genética
20.
J Bacteriol ; 187(19): 6862-6, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16166552

RESUMO

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.


Assuntos
DNA Polimerase beta/genética , Replicação do DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Mutagênese/genética , DNA Polimerase III/genética , DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Fenótipo
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