UC San Diego

In Escherichia coli, MutS initiates mismatch repair (MMR) by binding mispaired DNA. MutL, an intermediary protein, recognizes mispair-activated MutS and activates downstream MMR proteins. In eukaryotes MMR is similarly initiated by a MutS Homologue (MSH) complex called Msh2-Msh6 that binds to a MutL Homologue (MLH) complex, called Mlh1-Pms1, which activates downstream proteins. While the in vitro reconstitution of a MMR reaction using purified Escherichia coli proteins was described nearly twenty years ago (and recently using human proteins), the molecular mechanism of this process is not well understood. Accordingly, several models describe the coordination of MMR events. The studies described in this dissertation use various methods to explore the molecular mechanism of the initiation of MMR. A system was developed for studying MMR protein movement along DNA. This system was characterized using MSH and MLH complexes from Saccharomyces cerevisiae. In addition, an assay was developed to monitor ATP binding in the MSH proteins. These studies revealed that Msh2-Msh6 hydrolyzes ATP to bind mispaired DNA in an ADP-bound form. Mispair binding enables the Msh2-Msh6 to bind ATP in its high-affinity ATP binding site, but inhibits ATP hydrolysis allowing Msh2 to bind an additional ATP, yielding a dual ATP bound form, which is competent for sliding along DNA. Mutant MSH complexes defective for binding ATP in Msh2 failed to slide. The MSH-MLH ternary complex also appeared to slide, but its affinity for DNA ends confounded dissociation analysis. Interestingly, a dominant mutant Msh2-Msh6 complex interacted with Mlh1-Pms1, but failed to slide, suggesting that sliding is important for MMR in vivo. Lastly, small angle x-ray scattering of E. coli MutS and the crystal structure of its C-terminal 34 amino acids containing the tetramer-forming domain provide a model for full length MutS; further analysis revealed that stable dimers, but not tetramers are essential for MMR in vivo. Taken together, these results support a model of MMR initiation where a dimeric MSH complex recognizes the mispair, binds but does not hydrolyze ATP producing a conformational change that enables binding of the MLH complex and sliding along the DNA helix where downstream signaling can be initiated