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Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair.

Elez M, Radman M, Matic I - Nucleic Acids Res. (2012)

Bottom Line: We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci.A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold.Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Descartes, Sorbonne Paris Cité, Inserm Unit 1001, 75015 Paris, France. marina.elez@inserm.fr

ABSTRACT
Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.

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Related in: MedlinePlus

Functional fluorescent MutL and MutS co-localize on DNA mismatches. (A) Mutation frequency of mutL mutS deletion strain expressing fluorescently labeled MutS and MutL. Fluorescent images (taken with yellow and blue filters) and overlay image of mutH strain expressing (B) YFP-MutL and CFP-MutS or (C) YFP-MutS and CFP-MutL. Mean and standard error of the mean are presented in panel A. RifR stands for rifampicin resistant.
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gkr1298-F1: Functional fluorescent MutL and MutS co-localize on DNA mismatches. (A) Mutation frequency of mutL mutS deletion strain expressing fluorescently labeled MutS and MutL. Fluorescent images (taken with yellow and blue filters) and overlay image of mutH strain expressing (B) YFP-MutL and CFP-MutS or (C) YFP-MutS and CFP-MutL. Mean and standard error of the mean are presented in panel A. RifR stands for rifampicin resistant.

Mentions: Using GFP-MutL fusion, we and Walker's group showed previously that MutL forms MutS-dependent fluorescent foci on mismatches in growing E. coli and Bacillus subtilis cells (24,32). While the B. subtilis fluorescent MutL was non-functional, the E. coli one that we constructed conserved its function completely. For this study, we constructed YFP- and CFP-tagged MutL and MutS. To determine whether these fluorescent protein fusions are functional, we examined their ability to complement the deletions of mutS and mutL genes (Figure 1A and Supplementary Table S3). The strains deleted for mutL and mutS carrying the plasmid expressing YFP-MutL and CFP-MutS, or carrying the plasmid expressing CFP-MutL and YFP-MutL, exhibited the spontaneous mutagenesis levels of the wild-type strain (Figure 1A). On the other hand, the frequency of mutations of same mutL and mutS strains carrying only the empty vector was 200-fold higher than of the wild-type strain (Figure 1A).Figure 1.


Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair.

Elez M, Radman M, Matic I - Nucleic Acids Res. (2012)

Functional fluorescent MutL and MutS co-localize on DNA mismatches. (A) Mutation frequency of mutL mutS deletion strain expressing fluorescently labeled MutS and MutL. Fluorescent images (taken with yellow and blue filters) and overlay image of mutH strain expressing (B) YFP-MutL and CFP-MutS or (C) YFP-MutS and CFP-MutL. Mean and standard error of the mean are presented in panel A. RifR stands for rifampicin resistant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351158&req=5

gkr1298-F1: Functional fluorescent MutL and MutS co-localize on DNA mismatches. (A) Mutation frequency of mutL mutS deletion strain expressing fluorescently labeled MutS and MutL. Fluorescent images (taken with yellow and blue filters) and overlay image of mutH strain expressing (B) YFP-MutL and CFP-MutS or (C) YFP-MutS and CFP-MutL. Mean and standard error of the mean are presented in panel A. RifR stands for rifampicin resistant.
Mentions: Using GFP-MutL fusion, we and Walker's group showed previously that MutL forms MutS-dependent fluorescent foci on mismatches in growing E. coli and Bacillus subtilis cells (24,32). While the B. subtilis fluorescent MutL was non-functional, the E. coli one that we constructed conserved its function completely. For this study, we constructed YFP- and CFP-tagged MutL and MutS. To determine whether these fluorescent protein fusions are functional, we examined their ability to complement the deletions of mutS and mutL genes (Figure 1A and Supplementary Table S3). The strains deleted for mutL and mutS carrying the plasmid expressing YFP-MutL and CFP-MutS, or carrying the plasmid expressing CFP-MutL and YFP-MutL, exhibited the spontaneous mutagenesis levels of the wild-type strain (Figure 1A). On the other hand, the frequency of mutations of same mutL and mutS strains carrying only the empty vector was 200-fold higher than of the wild-type strain (Figure 1A).Figure 1.

Bottom Line: We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci.A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold.Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Descartes, Sorbonne Paris Cité, Inserm Unit 1001, 75015 Paris, France. marina.elez@inserm.fr

ABSTRACT
Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.

Show MeSH
Related in: MedlinePlus