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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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Dominant negative effect of roadblock MutHE56A over wild-type MutH. MutHE56A expression in the wild-type cells increases significantly (A) the percentage of cells with at least 1 MutL focus (B) the frequency of rifampicin-resistant mutants, compared to controls (P < 0.05, Wilcoxon test).
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gkr1298-F4: Dominant negative effect of roadblock MutHE56A over wild-type MutH. MutHE56A expression in the wild-type cells increases significantly (A) the percentage of cells with at least 1 MutL focus (B) the frequency of rifampicin-resistant mutants, compared to controls (P < 0.05, Wilcoxon test).

Mentions: We first tested how the expression of MutHE56A affects the frequency of MutL foci in wild-type and in mutH cells. We showed previously that the frequency of fluorescent MutL foci corresponds closely to the mutation frequency of different strains examined (24). We found that the production of MutHE56A increases significantly the frequency of MutL foci in the wild-type cells compared to controls (Figure 4A). We confirmed this result by a classical mutagenesis assay, i.e. we measured the frequency of appearance of spontaneous mutations conferring resistance to rifampicin in strains expressing MutHE56A and in the control wild-type strains (Figure 4B). The results show that expression of MutHE56A increased spontaneous mutagenesis.Figure 4.


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)

Dominant negative effect of roadblock MutHE56A over wild-type MutH. MutHE56A expression in the wild-type cells increases significantly (A) the percentage of cells with at least 1 MutL focus (B) the frequency of rifampicin-resistant mutants, compared to controls (P < 0.05, Wilcoxon test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1298-F4: Dominant negative effect of roadblock MutHE56A over wild-type MutH. MutHE56A expression in the wild-type cells increases significantly (A) the percentage of cells with at least 1 MutL focus (B) the frequency of rifampicin-resistant mutants, compared to controls (P < 0.05, Wilcoxon test).
Mentions: We first tested how the expression of MutHE56A affects the frequency of MutL foci in wild-type and in mutH cells. We showed previously that the frequency of fluorescent MutL foci corresponds closely to the mutation frequency of different strains examined (24). We found that the production of MutHE56A increases significantly the frequency of MutL foci in the wild-type cells compared to controls (Figure 4A). We confirmed this result by a classical mutagenesis assay, i.e. we measured the frequency of appearance of spontaneous mutations conferring resistance to rifampicin in strains expressing MutHE56A and in the control wild-type strains (Figure 4B). The results show that expression of MutHE56A increased spontaneous mutagenesis.Figure 4.

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