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Mol Microbiol. 2013 Nov;90(4):680-98. doi: 10.1111/mmi.12389. Epub 2013 Sep 16.

Trapping and visualizing intermediate steps in the mismatch repair pathway in vivo.

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1
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 North University Ave, Ann Arbor, MI, 48109-1048, USA.

Abstract

During mismatch repair, MutS is responsible for mismatch detection and the recruitment of MutL to the mismatch through a mechanism that is unknown in most organisms. Here, we identified a discrete site on MutS that is occupied by MutL in Bacillus subtilis. The MutL binding site is composed of two adjacent phenylalanine residues located laterally in an exposed loop of MutS. Disruption of this site renders MutS defective in binding MutL in vitro and in vivo, while also eliminating mismatch repair. Analysis of MutS repair complexes in vivo shows that MutS mutants defective in interaction with MutL are 'trapped' in a repetitive loading response. Furthermore, these mutant MutS repair complexes persist on DNA away from the DNA polymerase, suggesting that MutS remains loaded on mismatch proximal DNA awaiting arrival of MutL. We also provide evidence that MutS and MutL interact independent of mismatch binding by MutS in vivo and in vitro, suggesting that MutL can transiently probe MutS to determine if MutS is mismatch bound. Together, these data provide insights into the mechanism that MutS employs to recruit MutL, and the consequences that ensue when MutL recruitment is blocked.

PMID:
23998896
DOI:
10.1111/mmi.12389
[Indexed for MEDLINE]
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