The primary goal of this proposal is to elucidate the structure-function relationships that govern eukaryotic DNA mismatch repair. DNA mismatch repair (MMR) is the mechanism by which DNA synthesis errors are corrected post-replicatively, and it is central to the survival of all organisms. The proteins, MutS and MutL homologs, responsible for the initiation of mismatch repair are highly conserved throughout arid eukaryotes;while, the downstream repair events are less well conserved. MutS and MutL lomologs are dimeric proteins which contain both DNA binding and ATPase activities that are essential for MMR in vivo. MMR is initiated by MutS homologs binding to a mismatch. Subsequently, MutL homologs interact with the MutS homologs in an ATP-dependent manner and coordinate protein-protein interactions that signal excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. Recently eukaryotic bidirectional mismatch repair has been reconstituted in vitro using mismatch DNA containing a nick, and it requires the exonuclease, Exo1 (although others may be involved), the clamp loader protein, RFC, and clamp protein, PCNA, DNA polymerase 6, in addition to MutS and MutL proteins, and repair is enhanced in the presence of the single-stranded binding protein, RFA, and HMGB1. In humans, mutations in the MutS and MutL homologs are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with sporadic cancers. To understand how such mutations cause defects in mismatch repair, it is necessary to elucidate the molecular mechanisms of MMR and determine how mutations alter the mechanism. Biochemical studies indicate that different conformational states of the proteins and protein-DNA complexes are central to the regulation of MMR. To characterize these complexes, we will use atomic force microscopy (AFM) which provides an excellent method by which we can directly observe changes in conformational properties of such complexes. From a single set of AFM experiments, we can determine the binding affinity, specificity, and stoichiometry, as well as the conformational properties of the protein-DNA complexes. In addition, we can characterize conformational changes in single proteins and determine stoichiometries and association constants of protein-protein complexes. Finally, we can follow the dynamics of the protein-DNA complexes using solution imaging. As a complement to the AFM studies, we will use fluorescence anisotropy arid fluorescence resonance energy transfer (FRET) to characterize protein binding to DNA and protein-induced DNA bending in solution. Our long-term goal is to assemble complexes that are fully functional for DNA repair;however, in this study, we focus on the structure and function of several of the protein-protein and protein-DNA complexes that are involved in eukaryotic MMR.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Portnoy, Matthew
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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