application abstract) Mismatch repair plays a major role in genome stabilization, and available information indicates that the substrate specificity and mechanism of the reaction have been highly conserved during evolution. The Escherichia coli methyl-directed pathway, which is the best understood of the known mismatch repair systems, provides a paradigm for study of the mechanism of this complex reaction. Dr. Paul Modrich and his colleagues have identified eleven activities that are involved in mismatch repair, including MutS, MutL, MutH, DNA helicase II, and DNA polymerase III holoenzyme. This application addresses several features of the mechanism of this interesting reaction: (i) MutS and its eukaryotic homologs are responsible for mismatch recognition, but also possess a slow ATPase that is required for MutS function in mismatch repair. It is now clear that these proteins leave the mismatch in an ATP-dependent reaction to move along the helix contour, an effect that is believed to play an important role in the coupling of mismatch recognition to the recognition of a strand signal elsewhere on the helix that confers strand specificity on the reaction. However, the mechanism of this movement is controversial. They plan experiments that they hope will resolve this question with respect to the bacterial protein. (ii) In collaboration with the laboratory of Lorena Beese, they are pursuing structural analysis of bacterial MutS in order to clarify the basis of its ability to recognize mismatched base pairs. (iii) They showed that MutS and MutL load DNA helicase II at the strand break introduced by MutH, the key step in initiation of mismatch-provoked excision. They have also shown that MutL functions as an activator of the helicase. They hope to clarify the molecular basis of this activation. (iv) Several protein-DNA assemblies have been documented during the course of the methyl-directed reaction, although their nature has only been addressed in qualitative terms. They hope to establish the molecular composition of these complexes. This phase of the work will also address the basis of the specific requirement for DNA polymerase III holoenzyme in the repair synthesis step of the reaction.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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Wolfe, Paul B
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Duke University
Schools of Medicine
United States
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Modrich, Paul (2016) Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture). Angew Chem Int Ed Engl 55:8490-501
Pluciennik, Anna; Burdett, Vickers; Lukianova, Olga et al. (2009) Involvement of the beta clamp in methyl-directed mismatch repair in vitro. J Biol Chem 284:32782-91
Pluciennik, Anna; Modrich, Paul (2007) Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair. Proc Natl Acad Sci U S A 104:12709-13
Lopez de Saro, Francisco J; Marinus, Martin G; Modrich, Paul et al. (2006) The beta sliding clamp binds to multiple sites within MutL and MutS. J Biol Chem 281:14340-9
Iyer, Ravi R; Pluciennik, Anna; Burdett, Vickers et al. (2006) DNA mismatch repair: functions and mechanisms. Chem Rev 106:302-23
Bjornson, Keith P; Blackwell, Leonard J; Sage, Harvey et al. (2003) Assembly and molecular activities of the MutS tetramer. J Biol Chem 278:34667-73
Bjornson, Keith P; Modrich, Paul (2003) Differential and simultaneous adenosine di- and triphosphate binding by MutS. J Biol Chem 278:18557-62
Baitinger, Celia; Burdett, Vickers; Modrich, Paul (2003) Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex. J Biol Chem 278:49505-11
Viswanathan, M; Burdett, V; Baitinger, C et al. (2001) Redundant exonuclease involvement in Escherichia coli methyl-directed mismatch repair. J Biol Chem 276:31053-8
Blackwell, L J; Bjornson, K P; Allen, D J et al. (2001) Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis. J Biol Chem 276:34339-47

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