The Project (Mismatch Repair Interactions) integrates into the SBDR Program Project by focusing upon early responses to mutagenic mispaired bases and DNA adducts, including adducts made by chemotherapeutic agents. Mismatch repair is a major contributor to genome stability;defects in the mammalian pathway are associated with a strong predisposition to tumor development and inherited mutations in mismatch repair genes underlie one of the most prevalent inherited cancer susceptibility syndromes known. Despite the importance of this system in avoiding mutation, our understanding of its molecular nature is limited. The goals of this project are to establish the conformations and structures of multi-protein and multi-protein-DNA complexes that are the key intermediates in triggering the MutSa- and MutLa-dependent responses to mismatched base pairs and certain types of DNA damage. To accomplish this, our aims are four-fold: (1) The conformations and dynamics of multi-protein and multi-protein DNA assemblies involved in the initiation step of mismatch repair will be addressed by small angle X-ray scattering. These and other structural studies will exploit the high temporal resolution of the Structural Cell Biology (SCB) Synchrotron Beamline and the SCB Core. (2) The molecular basis for the recognition of base-base mispairs, insertion/deletion mispairs, and damaged DNA substrates will be addressed by X-ray crystallography. (3) Since the initiation of mismatch repair depends on assembly of multi-protein-DNA complexes (MutSa.MutLa.PCNA.DNA in the eukaryotic reaction) these multi-protein and multi-protein-DNA assemblies will be examined using X-ray crystallography. (4) The structural studies above will reveal residues at protein-protein interfaces as well as those that may be involved in conformational transitions;the significance of these residues will be subjected to biological validation by analysis of the phenotypic consequences of genetic alteration of these residues and by examination of selected mutant proteins at the biochemical level.
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