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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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Roadblock on strand discrimination site reduces the accumulation of MutL on DNA mismatches. (A) Percentage of cells with at least 1 MutL focus in mutH cells is not affected by MutHE56A expression (P > 0.05, Wilcoxon test). (B) Fluorescent images of mutH cells expressing (a) fluorescent MutL alone or (b) fluorescent MutL and MutHE56A mutant. (C) The mean focus fluorescence for 150 foci from Ba and for 150 foci from Bb is significantly different (P < 0.0001, Wilcoxon test). The mean cytoplasmic fluorescence for 90 cells from B(a) and 90 cells from B(b) is not significantly different (P > 0.05, Wilcoxon test). (D) The histograms of MutL foci fluorescence for cells from B(a) and B(b). Error bars in A and C indicate the standard error of the mean. n in A and C indicates the number of cells/foci examined.
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gkr1298-F5: Roadblock on strand discrimination site reduces the accumulation of MutL on DNA mismatches. (A) Percentage of cells with at least 1 MutL focus in mutH cells is not affected by MutHE56A expression (P > 0.05, Wilcoxon test). (B) Fluorescent images of mutH cells expressing (a) fluorescent MutL alone or (b) fluorescent MutL and MutHE56A mutant. (C) The mean focus fluorescence for 150 foci from Ba and for 150 foci from Bb is significantly different (P < 0.0001, Wilcoxon test). The mean cytoplasmic fluorescence for 90 cells from B(a) and 90 cells from B(b) is not significantly different (P > 0.05, Wilcoxon test). (D) The histograms of MutL foci fluorescence for cells from B(a) and B(b). Error bars in A and C indicate the standard error of the mean. n in A and C indicates the number of cells/foci examined.

Mentions: The expression of MutHE56A had no effect on the frequency of MutL foci in mutH cells compared to control cells (Figure 5A), arguing that these cells were still MMR deficient. On the other hand, the expression of MutHwt from a plasmid completely restored the MMR proficiency of the mutH cells. This was determined by measuring the frequency of YFP-MutL foci and the frequency of appearance of rifampicin resistant mutants (data not shown). These results are consistent with previous studies of mutation frequencies of mutH cells expressing MutHE56A or MutHwt (29).Figure 5.


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)

Roadblock on strand discrimination site reduces the accumulation of MutL on DNA mismatches. (A) Percentage of cells with at least 1 MutL focus in mutH cells is not affected by MutHE56A expression (P > 0.05, Wilcoxon test). (B) Fluorescent images of mutH cells expressing (a) fluorescent MutL alone or (b) fluorescent MutL and MutHE56A mutant. (C) The mean focus fluorescence for 150 foci from Ba and for 150 foci from Bb is significantly different (P < 0.0001, Wilcoxon test). The mean cytoplasmic fluorescence for 90 cells from B(a) and 90 cells from B(b) is not significantly different (P > 0.05, Wilcoxon test). (D) The histograms of MutL foci fluorescence for cells from B(a) and B(b). Error bars in A and C indicate the standard error of the mean. n in A and C indicates the number of cells/foci examined.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1298-F5: Roadblock on strand discrimination site reduces the accumulation of MutL on DNA mismatches. (A) Percentage of cells with at least 1 MutL focus in mutH cells is not affected by MutHE56A expression (P > 0.05, Wilcoxon test). (B) Fluorescent images of mutH cells expressing (a) fluorescent MutL alone or (b) fluorescent MutL and MutHE56A mutant. (C) The mean focus fluorescence for 150 foci from Ba and for 150 foci from Bb is significantly different (P < 0.0001, Wilcoxon test). The mean cytoplasmic fluorescence for 90 cells from B(a) and 90 cells from B(b) is not significantly different (P > 0.05, Wilcoxon test). (D) The histograms of MutL foci fluorescence for cells from B(a) and B(b). Error bars in A and C indicate the standard error of the mean. n in A and C indicates the number of cells/foci examined.
Mentions: The expression of MutHE56A had no effect on the frequency of MutL foci in mutH cells compared to control cells (Figure 5A), arguing that these cells were still MMR deficient. On the other hand, the expression of MutHwt from a plasmid completely restored the MMR proficiency of the mutH cells. This was determined by measuring the frequency of YFP-MutL foci and the frequency of appearance of rifampicin resistant mutants (data not shown). These results are consistent with previous studies of mutation frequencies of mutH cells expressing MutHE56A or MutHwt (29).Figure 5.

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