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病线圈(APC)基因在DNA修复活动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，其次是K-ras基因的顺序突变，在结直肠癌(DCC)和p53基因中缺失。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)和脱嘌呤/脱嘧啶核酸内切酶(APE)以及细胞周期激酶抑制蛋白p21相互作用。增殖细胞核抗原和APE在增殖细胞核抗原相互作用蛋白(PIP)盒上与APC相互作用，p21在APC的C末端相互作用。我们推测，野生型APC促进与增殖细胞核抗原和APE形成活性复合体，从而增加APE的活性，并通过长补丁(LP)-BER途径促进基本损伤的修复，从而防止正常结肠上皮细胞中基因突变的积累。截短APC的C末端区域导致p21结合丢失，p21在APC的PIP盒与增殖细胞核抗原结合，阻断了增殖细胞核抗原介导的APE活性和LP-BER。Lp-BER的降低导致突变的积累和结直肠肿瘤的发生和发展。为了验证这一假设，我们将：(1)在体外下拉和体内功能分析中确定APC与p21和BER蛋白的相互作用；(2)在存在或不存在基础DNA的情况下，对从细胞提取物中纯化的具有功能活性的BER复合体中APC/PCNA/APE/p21的组装进行表征；(3)确定野生型APC是否作为增殖细胞核抗原与基础DNA相互作用的招募因子，以及p21是否干扰突变的APC并降低这种活性；以及(4)确定p21介导的LP-BER降低是否与APE介导的3‘-5’外切酶活性增加有关。本项目将首次详细介绍野生型和突变型APC与p21共同参与BER活性的潜在分子机制，以及它在结直肠肿瘤发生和发展中的作用。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.