Involvement of APC in DNA Repair

描述（由申请人提供）：本提案的总体目标是确定腺瘤性息肉线圈（APC）基因在DNA修复活性和结直肠癌发展中的作用。APC基因突变是结直肠癌多步骤发展过程中最早发生的事件之一，随后是K-ras基因、DCC基因和p53基因的序列突变。APC基因的突变如何导致其他基因的突变尚不清楚。我们的初步数据表明，APC蛋白与DNA碱基切除修复（BER）蛋白、增殖细胞核抗原（PCNA）和无尿嘧啶/无嘧啶核酸内切酶（APE）以及细胞周期激酶抑制蛋白p21相互作用。PCNA和APE与APC在pna相互作用蛋白（PIP）盒上相互作用，p21在APC的c端区域相互作用。我们假设野生型APC水平促进PCNA和APE活性复合物的形成，从而增加APE活性，并通过长斑块(LP)-BER途径促进基础病变的修复，从而防止正常结肠上皮细胞中基因突变的积累。截断APC的c端区域导致p21失去结合，p21在APC的PIP-box上与PCNA结合，阻断了PCNA介导的APE活性和LP-BER。LP-BER的减少导致突变的积累和结直肠肿瘤发生的开始和进展。为了验证这一假设，我们将：(1)通过体外拉下和体内功能分析确定APC与p21和BER蛋白的相互作用；(2)在存在或不存在基本DNA的情况下，对从细胞提取物中纯化的功能活性BER复合物中APC/PCNA/APE/p21的组装进行了表征；(3)确定野生型APC是否作为PCNA与基本DNA相互作用的招募因子，以及p21是否干扰突变型APC并降低这种活性；(4)确定p21介导的LP-BER降低是否与ape介导的3‘ - 5’外切酶活性升高有关。该项目将首次详细阐述野生型和突变型APC与p21合作在BER活性及其在结直肠肿瘤发生的开始和进展中的作用的分子机制。这些研究结果将为开发一类新的预防结直肠癌的化疗药物奠定基础。

DESCRIPTION (provided by applicant): 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.