UC San Diego

DNA mismatch repair (MMR) is a conserved pathway that ensures replication fidelity by repairing errors that occur during replication of the genome. In eukaryotes MMR is initiated by two heterodimeric MutS homologue (MSH) complexes, Msh2-Msh3 and Msh2-Msh6, which recognize and bind mispairs and transmit signals to downstream components that effect repair. In this work we have investigated several aspects of the mechanisms by which the MSHs locate and recognize mispairs and initiate the downstream events in MMR. We have found that the N- terminal regions (NTRs) of Msh6 and Msh3 in Saccharomyces cerevisiae are long unstructured linkers that tether the MSH complexes to the sliding clamp PCNA. A second region of the Msh6 NTR is functionally partially redundant with the PCNA interacting region, and in the absence of the entire NTR MMR is severely impaired. The NTRs of Msh3 and Msh6 can be exchanged to create functional chimeric proteins, and the Msh6 NTR is functional when fused to Msh2. Here we also describe a chimeric S. cerevisiae MSH in which the mispair-binding domain of Msh6 is replaced by that of Msh3. The chimera is functional for MMR and the mispair-binding domain confers Msh3-like mispair recognition specificities. The chimera retains Msh6-like interactions with the MutL homologues, indicating that the mispair-binding domains and the type of mispair recognized do not contribute to the specificity of subsequent interactions. These findings suggest that the mispair- binding domains of Msh3 and Msh6 are modules that confer distinct mispair specificities but communicate signals of recognition to other domains in the same fashion. Additional studies described here demonstrate that a dominant negative mutation in the ATPase domain of Msh6 is sufficient to cause cancer in mice. However, cells from these mice do not show the increased resistance to drugs that is seen in MMR knock-out cells. Experiments in S. cerevisiae demonstrate that the mutation in these mice is equivalent to a previously described yeast mutation and a mutation in a human cancer case