Abstract 1172 Elucidation of Saccharomyces Cerevisiae Msh2-Msh6 and Holliday Junction Binding Interactions Via UV Photocrosslinking

The mismatch repair system is an evolutionarily conserved pathway that is vital in maintaining genomic integrity and cellular stability. It is involved in post-replication DNA repair and recombination processes and its inactivation is strongly linked to increased tumor development. The Saccharomyces cerevisiae MutSa or Msh2-Msh6 heterodimer is a post-replicative mismatch repair protein that recognizes and corrects single base mismatches and small insertion-deletion loops. Interestingly, Msh2-Msh6 binds DNA Holliday Junctions (HJ) with the same affinity as DNA mismatches; however, the specifics of this binding interaction and the key residues involved are relatively unknown. To investigate the orientation in which Msh2-Msh6 binds to HJs, the protein was photocrosslinked to DNA junction constructs with 5- bromouridine (BrU), which forms a stable crosslink to proteins upon exposure to 254nm UV light. Incorporation of BrU on different strands of a homologous junction did not reveal any binding asymmetry; although probe locations further from the junction center reduce crosslinking efficiency. The Msh6 subunit has been shown through crystallography to use a Phe-X-Glu motif for mismatch recognition. Crosslinking studies and fluorescence binding assays performed with mutant proteins suggest that this motif is not needed for junction recognition. To identify the residues important for the junction interaction, we are employing mass spectrometry to determine the protein residues crosslinked to BrU. In parallel, we have been incorporating a non-natural amino acid pBpA to photocrosslink the protein to the DNA and identify protein residues important for junction binding. Our preliminary results suggest Msh2-Msh6 may bind Holliday junctions in a different manner from DNA single base mismatches.