Dynamics of Mismatch and Alternative Excision-Dependent Repair in Replicating Bacillus subtilis DNA Examined Under Conditions of Neutral Selection
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SUBMITTER: Patlan-Vazquez A
PROVIDER: S-EPMC9280176 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an interaction with beta clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of the beta clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with beta clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and beta is the major molecular interaction facilitating focus formation and that beta clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with beta clamp.
| S-EPMC2350196 | biostudies-literature
Project description:The nucleotide excision repair (NER) and spore photoproduct lyase DNA repair pathways are major determinants of Bacillus subtilis spore resistance to UV radiation. We report here that a putative ultraviolet (UV) damage endonuclease encoded by ywjD confers protection to developing and dormant spores of B. subtilis against UV DNA damage. In agreement with its predicted function, a His(6)-YwjD recombinant protein catalyzed the specific incision of UV-irradiated DNA in vitro. The maximum expression of a reporter gene fusion to the ywjD opening reading frame occurred late in sporulation, and this maximal expression was dependent on the forespore-specific RNA polymerase sigma factor, ?(G). Although the absence of YwjD and/or UvrA, an essential protein of the NER pathway, sensitized developing spores to UV-C, this effect was lower when these cells were treated with UV-B. In contrast, UV-B but not UV-C radiation dramatically decreased the survival of dormant spores deficient in both YwjD and UvrA. The distinct range of lesions generated by UV-C and UV-B and the different DNA photochemistry in developing and dormant spores may cause these differences. We postulate that in addition to the UvrABC repair system, developing and dormant spores of B. subtilis also rely on an alternative excision repair pathway involving YwjD to deal with the deleterious effects of various UV photoproducts.
| S-EPMC3486385 | biostudies-literature
Project description:DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B. subtilis YwqL, a homolog of Endonuclease-V (EndoV), acts on a wider spectrum of deaminated bases but the factors that complete this pathway have remained elusive. Here, we report that a purified His6-YwqL (hereafter BsEndoV) protein had in vitro endonuclease activity against double-stranded DNAs containing a single uracil (U), hypoxanthine (Hx), xanthine (X) or an AP site. Interestingly, while BsEndoV catalyzed a single strand break at the second phosphodiester bond towards the 3'-end of the U and AP lesions, there was an additional cleavage of the phosphodiester bond preceding the Hx and X lesions. Remarkably, the repair event initiated by BsEndoV on Hx and X, was completed by a recombinant B. subtilis His6-DNA polymerase A (BsPolA), but not on BsEndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B. subtilis His6-ExoA (BsExoA) was necessary before completion of their repair by BsPolA. These results suggest the existence of a novel alternative excision repair pathway in B. subtilis that counteracts the genotoxic effects of base deamination. The presence of this novel pathway in vivo in B. subtilis was also supported by analysis of effects of single or multiple deletions of exoA, endoV and polA on spontaneous mutations in growing cells, and the sensitivity of growing wild-type and mutant cells to a DNA deaminating agent.
| S-EPMC6364969 | biostudies-literature
Project description:We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilisIMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.
| S-EPMC7148130 | biostudies-literature
Project description:Eukaryotic mismatch-repair (MMR) proteins MutSalpha and MutLalpha couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3' ends and 5' Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutSalpha ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutSalpha molecule per substrate. As the mismatch-nick DNA contour distance D in exogenous substrates increased from 0.26 to 0.98 kbp, initiation of excision in extracts decreased as D(-0.43) rather than the D(-1) to D(-2) predicted by some translocation or diffusion models. Virtually all excision was along the shorter (3'-5') nick-mismatch, even when the other (5'-3') path was less than twice as long. These observations argue against stochastically directed translocating/diffusing recognition complexes. The failure of mismatched DNA in trans to provoke excision of separate nicked homoduplexes argues against one-stage (concerted) triggering of excision initiation by recognition complexes acting through space. However, proteins associated with gapped DNA did appear to compete in trans with those in cis to mismatch-associated proteins. Thus, as in Escherichia coli, eukaryotic MMR may involve distinct initial-activation and excision-path-commitment stages.
| S-EPMC2175295 | biostudies-literature
Project description:The beta clamp is an essential replication sliding clamp required for processive DNA synthesis. The beta clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of beta clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49 degrees C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49 degrees C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the beta clamp, a common site occupied by proteins that bind the beta clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis beta clamp separate the role of the beta clamp in DNA replication from its role in MMR.
| S-EPMC2897676 | biostudies-literature
Project description:DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase ? (Pol?). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Pol? or Pol? active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Pol? were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Pol? were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Pol? is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Pol? that modulates the response to alkylation damage. These studies suggest that the Pol?/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.
| S-EPMC5689576 | biostudies-literature
Project description:Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.
| S-EPMC5630138 | biostudies-literature
Project description:The absence of base excision repair (BER) proteins involved in processing ROS-promoted genetic insults activates a DNA damage scanning (DisA)-dependent checkpoint event in outgrowing Bacillus subtilis spores. Here, we report that genetic disabling of transcription-coupled repair (TCR) or nucleotide excision repair (NER) pathways severely affected outgrowth of ΔdisA spores, and much more so than the effects of these mutations on log phase growth. This defect delayed the first division of spore's nucleoid suggesting that unrepaired lesions affected transcription and/or replication during outgrowth. Accordingly, return to life of spores deficient in DisA/Mfd or DisA/UvrA was severely affected by a ROS-inducer or a replication blocking agent, hydrogen peroxide and 4-nitroquinoline-oxide, respectively. Mutation frequencies to rifampin resistance (Rifr ) revealed that DisA allowed faithful NER-dependent DNA repair but activated error-prone repair in TCR-deficient outgrowing spores. Sequencing analysis of rpoB from spontaneous Rifr colonies revealed that mutations resulting from base deamination predominated in outgrowing wild-type spores. Interestingly, a wide range of base substitutions promoted by oxidized DNA bases were detected in ΔdisA and Δmfd outgrown spores. Overall, our results suggest that Mfd and DisA coordinate excision repair events in spore outgrowth to eliminate DNA lesions that interfere with replication and transcription during this developmental period.
| S-EPMC6182552 | biostudies-literature
Project description:Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.
| S-EPMC4812718 | biostudies-literature