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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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MutL amount on mismatches is independent of the intracellular concentration of MutL. No correlation between MutL focus fluorescence and cytoplasmic MutL fluorescence for (A) 131 foci without treatment (R2 = 0.007) and (B) 200 foci after 30-min treatment with rifamcipin (R2 = 0.004).
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gkr1298-F3: MutL amount on mismatches is independent of the intracellular concentration of MutL. No correlation between MutL focus fluorescence and cytoplasmic MutL fluorescence for (A) 131 foci without treatment (R2 = 0.007) and (B) 200 foci after 30-min treatment with rifamcipin (R2 = 0.004).

Mentions: Since, we established that MutL assembles in greater numbers on mismatches than MutS, we wondered if this was determined by the intracellular concentration of MutL. To examine this possibility, we quantified the cytoplasmic fluorescence of eGFP-MutL and examined its correlation with the fluorescence intensity of MutL foci. No correlation between these fluorescence intensities was seen (Figure 3A). We conclude that the cytoplasmic concentration of MutL does not determine its amount assembled on mismatches.Figure 3.


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)

MutL amount on mismatches is independent of the intracellular concentration of MutL. No correlation between MutL focus fluorescence and cytoplasmic MutL fluorescence for (A) 131 foci without treatment (R2 = 0.007) and (B) 200 foci after 30-min treatment with rifamcipin (R2 = 0.004).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1298-F3: MutL amount on mismatches is independent of the intracellular concentration of MutL. No correlation between MutL focus fluorescence and cytoplasmic MutL fluorescence for (A) 131 foci without treatment (R2 = 0.007) and (B) 200 foci after 30-min treatment with rifamcipin (R2 = 0.004).
Mentions: Since, we established that MutL assembles in greater numbers on mismatches than MutS, we wondered if this was determined by the intracellular concentration of MutL. To examine this possibility, we quantified the cytoplasmic fluorescence of eGFP-MutL and examined its correlation with the fluorescence intensity of MutL foci. No correlation between these fluorescence intensities was seen (Figure 3A). We conclude that the cytoplasmic concentration of MutL does not determine its amount assembled on mismatches.Figure 3.

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