The overall goal of this proposal is to determine the role of the adenomatous polyposis coil (APC) gene in DNA repair activity and the development of colorectal cancer. Mutation of the APC gene is one of the earliest events in the multistep process of the development of colorectal cancer, and is followed by sequential mutations in K-ras, deleted in colorectal cancer (DCC) and p53 genes. How mutation of the APC gene leads to the mutation of other genes is unclear. Our preliminary data indicate that the APC protein interacts with the DNA base excision repair (BER) proteins, proliferating cell nuclear antigen (PCNA) and apurinic/apyrimidinic endonuclease (APE), and the cell cycle kinase inhibitor protein, p21. PCNA and APE interact with APC at the PCNA-interacting protein (PIP)-box and p21 interacts at the C-terminal region of APC. We hypothesize that wild-type levels of APC promote formation of an active complex with PCNA and APE, which increases APE activity and facilitates repair of abasic lesions through a long-patch (LP)-BER pathway thereby preventing accumulation of gene mutations in normal colonic epithelial cells. Truncation of the C-terminal region of APC leads to loss of p21 binding and the p21 binds with PCNA at the PIP-box of APC, blocking PCNA-mediated APE activity and LP-BER. The decrease in LP-BER results in the accumulation of mutations and the initiation and progression of colorectal tumorigenesis. To test this hypothesis, we will: (1) Determine the interactions of APC with p21 and BER proteins in in vitro pull-down and in vivo functional assays; (2) Characterize APC/PCNA/APE/p21 assembly in the functionally active BER complexes purified from the cellular extracts in the presence or absence of abasic DNA; (3) Determine whether the wild-type APC acts as a recruitment factor for PCNA interaction with abasic DNA and whether p21 interferes with mutant APC and decreases this activity; and (4) Determine whether the p21-mediated decrease in LP-BER is associated with APE-mediated increase in 3' - 5' exonuclease activity. This project will, for the first time, detail the molecular mechanisms underlying the function of wild-type and mutant APC in collaboration with p21 in BER activity and its role in the initiation and progression of colorectal tumorigenesis. The findings of these studies will lay the basis for the development of a new class of chemotherapeutic agents for the prevention of colorectal cancers. ? ?
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