The objectives of this application are two-fold: 1. The recognition and processing of mismatched DNA base pairs contributes to the fidelity of chromosome replication and may also be responsible for certain marker effects in recombination. We have developed a cell-free E. coli system which supports methyl-directed mismatch correction in vitro, and have obtained all the proteins known to be required for this process (products of mutH, mutL, mutS, and uvrD genes) in near pure form. Since these proteins are not sufficient to mediate the reaction, we will isolate other required components with the aim of reconstituting the reaction in a defined system. This is a prerequisite for a major aim of the work, namely to establish the biochemical mechanism by which this complex reaction occurs. Our second goal with this system is to exploit its ability to recognize mispaired bases in attempts to develop a reagent system for the detection and localization of point mutations in vitro. 2. The second major objective of the proposed work is to further elucidate the molecular mechanisms involved in specific DNA sequence recognition and catalysis by EcoRI restriction and modification enzymes. Our efforts in this respect will be along two lines. Utilizing kinetic and binding methods, we will try to further delineate the mechanisms of DNA search and site-specific catalysis by these enzymes. Secondly, we will continue ongoing molecular genetic experiments, the aim of which is to relate particular amino acid residues to functional activities of the two proteins.
| 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 |
| 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 |
| 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 |
| 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 |
| Burdett, V; Baitinger, C; Viswanathan, M et al. (2001) In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair. Proc Natl Acad Sci U S A 98:6765-70 |
| 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 |
| Spampinato, C; Modrich, P (2000) The MutL ATPase is required for mismatch repair. J Biol Chem 275:9863-9 |
| Bjornson, K P; Allen, D J; Modrich, P (2000) Modulation of MutS ATP hydrolysis by DNA cofactors. Biochemistry 39:3176-83 |
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