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DNA REPAIR ACTIVITIES OF HUMAN ABASIC ENDONUCLEASE

人碱基核酸内切酶的 DNA 修复活性

基本信息

批准号：
6350320
负责人：
DANIEL BARSKY
金额：
$ 31.97万
依托单位：
UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-04-01 至 2003-11-30
项目状态：
已结题

项目摘要

Genetic material is subject to spontaneous and mutagen-induced modifications that contribute to the process of carcinogenesis. The most frequently formed damages are likely those produced by free radical attack of DNA. Free radicals, which include superoxide and hydroxyl radicals, are generated during normal oxygen metabolism and from exposure to anti-cancer agents such as bleomycin and ionizing radiation. Mutagenic or lethal oxidative DNA damages include sites of base loss (abasic or AP sites) and 3'-obstructive termini (3'-phosphate or 3'- phosphoglycolate). Enzymes that initiate the repair of AP sites and 3'- damages have been identified in all organisms studied to date, from bacteria to humans, and are a central component of base excision DNA repair. The predominant AP endonuclease in mammals is Ape/Hap1/Ref-1, but unlike other AP endonucleases, Ape possesses only a minor 3'-repair activity. Although many of the general features of Ape have been elucidated, the specifics of its recognition and binding to abasic site-containing DNA are not well understood. Using biochemical (e.g. footprinting, protein-DNA crosslinking, and site-directed mutagenesis) and structural (Nuclear Magnetic Resonance and Circular Dichroism) approaches, we intend to unravel the details of the Ape-DNA interaction and to identify the critical points of contact. In addition, we will employ computer modeling to define the structural features of DNA substrates utilized in these and other studies to uncover aspects of DNA that are important for recognition and incision. Elucidating the molecular detail of the Ape-DNA interaction may permit the design of effective inhibitor substrates, which may aid cancer treatment regimes and Ape-DNA structural studies, or of a dominant-negative Ape protein, which would help to clarify the biological importance of this enzyme. Since Ape displays a relatively poor 3'-diesterase activity in vitro, particularly at double-strand break ends, the biological role of this activity is unclear. A recent study reporting an interaction between Ape and Ku could suggest that these proteins act in cooperation to efficiently process 3'-damages, and may indicate a role for Ape in non- homologous recombination (i.e. DNA end-joining). We intend to characterize this interaction biochemically and determine whether Ku influences the 3'-repair capacity of Ape. We intend to identify and characterize other nucleases or accessory factors that are involved in the processing of 3'-oxidative damages. The studies described within will go a long way towards defining the repair activities of Ape and will help understand the intracacies of two critical DNA repair pathways.

遗传物质易受自发和诱变剂诱导的导致致癌过程的修饰。最常形成的损伤可能是自由基 DNA攻击自由基，包括超氧化物和羟基自由基，产生于正常的氧代谢过程中，暴露于抗癌剂如博来霉素和电离辐射。致突变性或致死性氧化性DNA损伤包括碱基丢失位点（脱碱基或AP位点）和3 '-阻塞性末端（3'-磷酸或3 '-磷酸）磷酸乙醇酸盐）。启动AP位点和3'- 迄今为止，在所有研究的生物体中都发现了损害，细菌对人类，是一个核心组成部分的碱基切除DNA 修复. 哺乳动物中主要的AP核酸内切酶是Ape/Hap 1/Ref-1，但与其他AP内切酶不同，Ape仅具有较小的3 '-修复，活动尽管Ape的许多一般特征已经被阐明，其识别和结合脱碱基的细节含位点DNA还没有被很好地理解。使用生物化学（例如，足迹法、蛋白质-DNA交联和定点诱变）核磁共振和圆二色性（NMR and Circular Dichroism）我们打算解开Ape-DNA相互作用的细节，并确定关键的接触点。此外，我们会使用计算机建模来定义DNA的结构特征在这些和其他研究中使用的底物，以揭示DNA 这对识别和切开很重要阐明 Ape-DNA相互作用的分子细节可能允许设计有效的抑制剂底物，可帮助癌症治疗方案和猿DNA结构研究，或显性阴性猿蛋白的研究，这将有助于阐明这种酶的生物学重要性。由于Ape在体外显示出相对较差的3 ′-二酯酶活性，特别是在双链断裂末端，这种生物学作用活动不清楚。最近的一项研究报告了 Ape和Ku可能认为这些蛋白质协同作用，有效地处理3'-损伤，并可能表明猿在非同源重组（即DNA末端连接）。我们打算以生物化学的方式表征这种相互作用，并确定Ku 影响Ape的3 '-修复能力。我们打算确定和表征参与的其他核酸酶或辅助因子， 3'-氧化损伤的处理。其中描述的研究将大大有助于确定猿类的修复活动，将有助于理解两个关键的DNA修复的复杂性，途径。