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1.
Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.  相似文献   

2.
The Escherichia coli beta sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the beta clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant beta clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant beta clamp protein (beta159) is impaired for interactions with the alpha catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the beta clamp might help to coordinate protein traffic at the replication fork.  相似文献   

3.
The Escherichia coli dnaN159 allele encodes a mutant form of the beta-sliding clamp (beta159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the beta clamp stimulates the processivity of Pol I in vitro and that beta159 is impaired for this activity. The reduced ability of beta159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the beta clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 DeltapolA strain.  相似文献   

4.
Escherichia coli strains expressing the mutant beta159-sliding clamp protein (containing both a G66E and a G174A substitution) are temperature sensitive for growth and display altered DNA polymerase (pol) usage. We selected for suppressors of the dnaN159 allele able to grow at 42 degrees C, and identified four intragenic suppressor alleles. One of these alleles (dnaN780) contained only the G66E substitution, while a second (dnaN781) contained only the G174A substitution. Genetic characterization of isogenic E. coli strains expressing these alleles indicated that certain phenotypes were dependent upon only the G174A substitution, while others required both the G66E and G174A substitutions. In order to understand the individual contributions of the G66E and the G174A substitution to the dnaN159 phenotypes, we utilized biochemical approaches to characterize the purified mutant beta159 (G66E and G174A), beta780 (G66E) and beta781 (G174A) clamp proteins. The G66E substitution conferred a more pronounced effect on pol IV replication than it did pol II or pol III, while the G174A substitution conferred a greater effect on pol III and pol IV than it did pol II. Taken together, these findings indicate that pol II, pol III and pol IV interact with distinct, albeit overlapping surfaces of the beta clamp.  相似文献   

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6.
Sutton MD  Duzen JM 《DNA Repair》2006,5(3):312-323
Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.  相似文献   

7.
Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.  相似文献   

8.
DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.  相似文献   

9.
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.  相似文献   

10.
The Escherichia coli beta 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 beta clamp (beta159), 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., DeltarecR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., DeltapolB or DeltaumuDC). 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.  相似文献   

11.
DNA replication machineries tend to stall when confronted with damaged DNA template sites, causing the biochemical equivalent of a major 'train wreck'. A newly discovered bacterial DNA polymerase, Escherichia coli Pol V, acting in conjunction with the RecA protein, can exchange places with the stalled replicative Pol III core and catalyse 'error-prone' translesion synthesis. In contrast to Pol V-catalysed 'brute-force, sloppier copying', another SOS-induced DNA polymerase, Pol II, plays a pivotal role in an 'error-free', replication-restart DNA repair pathway and probably involves RecA-mediated homologous recombination.  相似文献   

12.
DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.  相似文献   

13.
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.  相似文献   

14.
Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.  相似文献   

15.
Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.  相似文献   

16.
Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N2-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N2-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.  相似文献   

17.
Bunting KA  Roe SM  Pearl LH 《The EMBO journal》2003,22(21):5883-5892
Y-family DNA polymerases can extend primer strands across template strand lesions that stall replicative polymerases. The poor processivity and fidelity of these enzymes, key to their biological role, requires that their access to the primer-template junction is both facilitated and regulated in order to minimize mutations. These features are believed to be provided by interaction with processivity factors, beta-clamp or proliferating cell nuclear antigen (PCNA), which are also essential for the function of replicative DNA polymerases. The basis for this interaction is revealed by the crystal structure of the complex between the 'little finger' domain of the Y-family DNA polymerase Pol IV and the beta-clamp processivity factor, both from Escherichia coli. The main interaction involves a C-terminal peptide of Pol IV, and is similar to interactions seen between isolated peptides and other processivity factors. However, this first structure of an entire domain of a binding partner with an assembled clamp reveals a substantial secondary interface, which maintains the polymerase in an inactive orientation, and may regulate the switch between replicative and Y-family DNA polymerases in response to a template strand lesion.  相似文献   

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