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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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The stoichiometry of MutL and MutS on DNA mismatches. (A) The mean cytoplasmic fluorescence and the mean fluorescence of foci for 108 cells with YFP-MutL and CFP-MutS foci from 1B and 95 cells with YFP-MutS and CFP-MutL foci from 1C. (B) The histogram of the ratio of MutL focus fluorescence to co-localized MutS focus fluorescence for 203 pairs of co-localized MutL and MutS foci from A. (C) No correlation between the fluorescence intensity of co-localized foci of YFP-MutL and CFP-MutS (R2 = 0.09) and (D) YFP-MutS and CFP-MutL (R2 = 0.08). Error bars in A indicate standard error of the mean, N in B stands for number, a.u. in A, C and D stands for arbitrary units and fluo in C and D stands for fluorescence.
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gkr1298-F2: The stoichiometry of MutL and MutS on DNA mismatches. (A) The mean cytoplasmic fluorescence and the mean fluorescence of foci for 108 cells with YFP-MutL and CFP-MutS foci from 1B and 95 cells with YFP-MutS and CFP-MutL foci from 1C. (B) The histogram of the ratio of MutL focus fluorescence to co-localized MutS focus fluorescence for 203 pairs of co-localized MutL and MutS foci from A. (C) No correlation between the fluorescence intensity of co-localized foci of YFP-MutL and CFP-MutS (R2 = 0.09) and (D) YFP-MutS and CFP-MutL (R2 = 0.08). Error bars in A indicate standard error of the mean, N in B stands for number, a.u. in A, C and D stands for arbitrary units and fluo in C and D stands for fluorescence.

Mentions: When the number of mismatches increases in the cell, MutL but not MutS becomes limiting (18–22). MutL could become limiting during MMR if the stoichiometry of the mismatch repair reaction is such that more MutL than MutS proteins are needed in the repair of a mismatch. This hypothesis predicts that the MutS and MutL foci co-localized on the mismatches should contain a greater amount of MutL than MutS protein. To compare the relative amount of MutS and MutL proteins per mismatch, we analyzed the fluorescence intensity of the co-localized MutS and MutL foci. First, we identified a MutL focus and the coincident MutS focus, and then we measured their respective fluorescent intensities. We performed these experiments with the combinations of YFP-MutL and CFP-MutS as well as with CFP-MutL and YFP-MutS protein fusions (Figure 1B and C). In both cases, the fluorescence intensity of MutL foci was higher compared to the intensity of MutS foci (Figure 2A). The average ratio of MutL foci fluorescence to co-localized MutS foci fluorescence was 2.7 (Figure 2B). These differences are not due to differences in the cytoplasmic fluorescence that varies very little among cells carrying differently labeled MutS and MutL proteins (Figure 2A).Figure 2.


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)

The stoichiometry of MutL and MutS on DNA mismatches. (A) The mean cytoplasmic fluorescence and the mean fluorescence of foci for 108 cells with YFP-MutL and CFP-MutS foci from 1B and 95 cells with YFP-MutS and CFP-MutL foci from 1C. (B) The histogram of the ratio of MutL focus fluorescence to co-localized MutS focus fluorescence for 203 pairs of co-localized MutL and MutS foci from A. (C) No correlation between the fluorescence intensity of co-localized foci of YFP-MutL and CFP-MutS (R2 = 0.09) and (D) YFP-MutS and CFP-MutL (R2 = 0.08). Error bars in A indicate standard error of the mean, N in B stands for number, a.u. in A, C and D stands for arbitrary units and fluo in C and D stands for fluorescence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1298-F2: The stoichiometry of MutL and MutS on DNA mismatches. (A) The mean cytoplasmic fluorescence and the mean fluorescence of foci for 108 cells with YFP-MutL and CFP-MutS foci from 1B and 95 cells with YFP-MutS and CFP-MutL foci from 1C. (B) The histogram of the ratio of MutL focus fluorescence to co-localized MutS focus fluorescence for 203 pairs of co-localized MutL and MutS foci from A. (C) No correlation between the fluorescence intensity of co-localized foci of YFP-MutL and CFP-MutS (R2 = 0.09) and (D) YFP-MutS and CFP-MutL (R2 = 0.08). Error bars in A indicate standard error of the mean, N in B stands for number, a.u. in A, C and D stands for arbitrary units and fluo in C and D stands for fluorescence.
Mentions: When the number of mismatches increases in the cell, MutL but not MutS becomes limiting (18–22). MutL could become limiting during MMR if the stoichiometry of the mismatch repair reaction is such that more MutL than MutS proteins are needed in the repair of a mismatch. This hypothesis predicts that the MutS and MutL foci co-localized on the mismatches should contain a greater amount of MutL than MutS protein. To compare the relative amount of MutS and MutL proteins per mismatch, we analyzed the fluorescence intensity of the co-localized MutS and MutL foci. First, we identified a MutL focus and the coincident MutS focus, and then we measured their respective fluorescent intensities. We performed these experiments with the combinations of YFP-MutL and CFP-MutS as well as with CFP-MutL and YFP-MutS protein fusions (Figure 1B and C). In both cases, the fluorescence intensity of MutL foci was higher compared to the intensity of MutS foci (Figure 2A). The average ratio of MutL foci fluorescence to co-localized MutS foci fluorescence was 2.7 (Figure 2B). These differences are not due to differences in the cytoplasmic fluorescence that varies very little among cells carrying differently labeled MutS and MutL proteins (Figure 2A).Figure 2.

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