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

Nucleic Acids Res. 2012 May;40(9):3929-38. doi: 10.1093/nar/gkr1298. Epub 2012 Jan 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / analysis*
  • Adenosine Triphosphatases / genetics
  • Base Pair Mismatch*
  • DNA / chemistry
  • DNA / metabolism
  • DNA Mismatch Repair*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / analysis*
  • Escherichia coli Proteins / genetics
  • Fluorescent Dyes
  • Luminescent Proteins / genetics
  • MutL Proteins
  • MutS DNA Mismatch-Binding Protein / analysis*
  • MutS DNA Mismatch-Binding Protein / genetics
  • Recombinant Fusion Proteins / analysis

Substances

  • Escherichia coli Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • MutL protein, E coli
  • Recombinant Fusion Proteins
  • DNA
  • Adenosine Triphosphatases
  • MutL Proteins
  • MutS DNA Mismatch-Binding Protein
  • MutS protein, E coli