Mismatch repair (MMR) is responsible for the removal of incorrectly paired bases resulting from replication errors. Failure to remove such errors can lead to genomic instability and cancer. Eukaryotic components have been identified in yeast based on the """"""""mutator phenotypes"""""""" due to gene deletion. Consistent with their roles in MMR, mutations in three of these human proteins (hMsh2, hMlhl, and hMsh6) are associated with hereditary nonpolyposis colorectal carcinoma (HNPCC). Notably missing from the eukaryotic MMR proteins identified to date are clear homologues of excision nucleases. This is due to low sequence similarity between prokaryotic and eukaryotic proteins and redundancy of the downstream components in this pathway. The finding that four excision enzymes are involved in MMR in E. coli suggests that multiple redundant nucleases may likewise participate in eukaryotic MMR. The quadruple null mutant E. coli strain is sensitive to 2-aminopurine, which provides a powerful functional screening tool to search the eukaryotic excision nucleases in the eukaryotic cell genomes. I will screen the complete yeast cDNA library using this tool in Specific Aim 1. The findings of several studies including our own work suggest that one of these excision nucleases is exonuclease 1. The nuclease protein interacts with major MMR proteins (Msh2, Mlhl, Msh3, and Msh6). Single deletion of S. cerevisiae EX01 results in a weak mutator phenotype while it is synergistic with mutatants of other established MMR genes. Our recent study indicated that functional alterations are associated with point mutations in the human EX01 gene identified in HNPCC. Work has been proposed to knock these mutations into mouse genome to test a hypothesis that the functional alterations in hEXO1 gene will cause colorectal tumorigenesis (Specific Aim 3). I have additionally found that the budding yeast ORF YJR001W shares sequence homology with E. coli excision nucleases and deletion of this ORF along with EX01 results in a synergistic effect on mutation frequency. We will characterize the function of Yjr001w in MMR (Specific Aim 2). A combination of approaches including in vitro biochemical and in vivo E. coli, yeast, and mouse genetic analyses may lead to a breakthrough in the bottleneck of this field. The obtained results should yield important insights into the molecular mechanisms of MMR as well as providing new target molecules for the detection and treatment of cancer and other diseases.
