<scp>DnaN</scp> clamp zones provide a platform for spatiotemporal coupling of mismatch detection to <scp>DNA</scp> replication

Lenhart, Justin S.; Sharma, Anil; Hingorani, Manju; Simmons, Lyle A.

doi:10.1111/mmi.12115

Cited by 39 publications

(78 citation statements)

References 98 publications

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“…To test whether rNMP incorporation may contribute to MMR in B. subtilis, we measured spontaneous mutation rates (28)(29)(30). We find that deletion of rnhB results in a 2.4-fold increase in mutation rate, whereas loss of rnhC appears nonmutagenic with a 1.3-fold increase relative to WT (P = 0.03 and 0.2, respectively).…”

Section: Resultsmentioning

confidence: 99%

Cost of rNTP/dNTP pool imbalance at the replication fork

Yao

Schroeder

Yurieva

et al. 2013

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

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101

188

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The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and e. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.T he structures of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) differ by a single atom, yet each must be distinguished by DNA and RNA polymerases (Pols). This is more challenging for DNA Pols because the intracellular concentration of rNTPs is 10-100-fold higher than that of dNTPs (1-3). DNA is more stabile than RNA, and rNMP residues in DNA could lead to spontaneous strand breaks. Hence, it is important to genomic integrity that DNA Pols exclude rNMPs from incorporation into the genome (1, 4).Structural studies reveal that DNA Pols distinguish ribo and deoxyribo sugars via a "steric gate," in which a bulky residue or main chain atom sterically occludes binding of the ribo 2′OH (5, 6). However, the single-atom difference between rNTPs and dNTPs imposes an upper limit to sugar recognition, and thus DNA Pols incorporate rNMPs at a low frequency. For example, studies in yeast demonstrate that rNMPs are incorporated in vivo, and studies in vitro demonstrate a frequency of rNMP incorporation predicting that 10,000 rNMPs or more may be incorporated each replication cycle (2,7,8). Given their abundant incorporation, there are probably multiple pathways to remove rNMPs, as their persistence is associated with genomic instability in yeast (7, 9).The current study reconstitutes the Escherichia coli replisome and examines the cost of rNTP/dNTP nucleotide pool imbalance on the frequency of rNMP incorporation and the rate of fork progression. We find that a nucleotide pool imbalance slows the ...

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Section: Resultsmentioning

confidence: 99%

Cost of rNTP/dNTP pool imbalance at the replication fork

Yao

Schroeder

Yurieva

et al. 2013

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

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101

188

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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%

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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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“…Earlier efforts by the Walker and Grossman laboratories (9,10) to localize MutS in live B. subtilis cells exploited a MutS-green fluorescent protein fusion (MutS-GFP). Although this work supports the idea that MMR is replication coupled, recent work demonstrated that only ∼10% of cells display detectable MutS-GFP foci, with only approximately one-half of these colocalizing with the replication fork (11). Thus, the extent to which the MutS-replication fork association contributes to MMR function is unclear.…”

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confidence: 49%

How MutS finds a needle in a haystack

Sutton

2015

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

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DnaN clamp zones provide a platform for spatiotemporal coupling of mismatch detection to DNA replication

Cited by 39 publications

References 98 publications

Cost of rNTP/dNTP pool imbalance at the replication fork

Cost of rNTP/dNTP pool imbalance at the replication fork

MutS2 Promotes Homologous Recombination in Bacillus subtilis

How MutS finds a needle in a haystack

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