Trapping and visualizing intermediate steps in the mismatch repair pathway <i>in vivo</i>

Lenhart, Justin S.; Pillon, Monica C.; Guarné, Alba; Simmons, Lyle A.

doi:10.1111/mmi.12389

Cited by 30 publications

(34 citation statements)

References 63 publications

(138 reference statements)

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“…Our previous research investigating MutS1 has shown that B. subtilis MutS1-GFP forms foci that are recruited to the site of DNA synthesis (25)(26)(27)(28)48). Further, homologous recombination proteins and nucleotide excision repair proteins have been shown to form foci and localize to the nucleoid, respectively, following MMC treatment (44,49).…”

Section: Together These Results Indicate That the C-terminal Smr Dommentioning

confidence: 99%

“…For live imaging experiments, a plate grown overnight at 30°C was washed using S7 50 minimal medium supplemented with 2% glucose essentially as described previously (25,26,59). The OD 600 was measured and cells were diluted to an OD 600 of 0.1.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were then placed on 1% agarose pads made of 1ϫ Spizizen's salts (53,54). Cells were imaged using an Olympus BX61 microscope as described previously (25,26,59). …”

Section: Methodsmentioning

confidence: 99%

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MutS2 Promotes Homologous Recombination in Bacillus subtilis

Burby

Simmons

2017

J Bacteriol

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Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Grampositive bacterium Bacillus subtilis. In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis. We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.IMPORTANCE Cells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination, providing a new function for MutS2. We found that cells lacking mutS2 are sensitive to DNA damage that requires homologous recombination for repair and have reduced transformation efficiency. Further analysis indicates that the C-terminal Smr domain requires the N-terminal portion of MutS2 for function in vivo. Moreover, we show that a mutS2 deletion is additive with a recU deletion, suggesting that these proteins have a redundant function in homologous recombination. Together, our study shows that MutS2 proteins have adapted different functions that impact recombination.

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Section: Together These Results Indicate That the C-terminal Smr Dommentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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MutS2 Promotes Homologous Recombination in Bacillus subtilis

Burby

Simmons

2017

J Bacteriol

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“…Shown are bar graphs representing the spontaneous mutation rate (Ϯ95% confidence intervals) using the MSS maximum-likelihood method, as described previously (31,63,(67)(68)(69). (A) Number of mutations per generation for wild-type strain PY79 and the ⌬recD2 and ⌬mutSL strains from at least 50 independent cultures plated on rifampin; (B) number of mutations per generation of the PY79, ⌬recD2, and ⌬mutSL strains from at least 20 independent cultures plated on trimethoprim; (C) number of mutations per generation of the indicated strains plated on rifampin.…”

Section: Fig 2 Deletion Of Recd2 Increases Spontaneous Mutagenesis Inmentioning

confidence: 99%

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

et al. 2014

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bDNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ⌬recD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ⌬recD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5=-3= helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.

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“…After recognizing a mismatch, MutS exchanges bound ADP for ATP and recruits MutL (1, 15). This MutS-MutL interaction is required for MutS to release the mismatch (16). MutS[K608A], which lacks detectable nucleotide binding activity, failed to localize to the replication fork, demonstrating that, at a minimum, mismatch detection by MutS requires its interaction with both β-clamp and nucleotide cofactor.…”

mentioning

confidence: 99%

How MutS finds a needle in a haystack

Sutton

2015

Proc. Natl. Acad. Sci. U.S.A.

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Trapping and visualizing intermediate steps in the mismatch repair pathway in vivo

Cited by 30 publications

References 63 publications

MutS2 Promotes Homologous Recombination in Bacillus subtilis

MutS2 Promotes Homologous Recombination in Bacillus subtilis

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

How MutS finds a needle in a haystack

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