DNA REPAIR ACTIVITIES OF HUMAN ABASIC ENDONUCLEASE

遗传物质受到自发和诱变剂的影响 有助于癌变过程的修饰。最多的 经常形成的损伤很可能是由自由基造成的 DNA的攻击。自由基，包括超氧化物和羟基 自由基，是在正常的氧气代谢过程中产生的， 接触抗癌药物，如博莱霉素和电离辐射。 致突变或致命性氧化DNA损伤包括碱基丢失部位 (碱性或AP位点)和3‘-阻塞性末端(3’-磷酸或3‘- 磷酸乙醇酸盐)。启动AP部位修复的酶和3‘- 到目前为止，已经在所有研究的生物体中发现了损害，来自 细菌对人类的影响，是碱基切除DNA的中心组成部分 修理。哺乳动物中主要的AP内切酶是Ape/Hap1/Ref-1， 但与其他AP核酸内切酶不同的是，APE只有一个微小的3‘-修复 活动。尽管类人猿的许多一般特征一直是 阐明了其识别和结合ABASIC的特异性 含有位点的DNA还没有被很好地理解。使用生化(例如 足迹、蛋白质-DNA交联和定点突变) 和结构(核磁共振和圆二色谱) 方法，我们打算揭开猿-DNA相互作用的细节 并确定关键的接触点。此外，我们还将 使用计算机建模来确定DNA的结构特征 在这些和其他研究中使用的底物，以揭示DNA的某些方面 这对识别和切割很重要。澄清 Ape-DNA相互作用的分子细节可能允许设计 有效的抑制物底物，可能有助于癌症治疗方案 以及Ape-DNA结构研究，或显性阴性Ape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在体外表现出相对较差的3‘-二酯酶活性， 尤其是在双链断裂末端，这一生物作用 活动情况尚不清楚。最近的一项研究报告了一种相互作用 Ape和Ku可能认为这些蛋白质协同作用于 有效地处理3‘-损害，并可能指示APE在非 同源重组(即DNA末端连接)。我们打算 用生化方法描述这种相互作用，并确定Ku 影响类人猿的3‘-修复能力。我们打算确定和 描述其他核酸酶或辅助因子参与的 3‘-氧化损伤的处理。中所述的研究 将对定义Ape和Ape的维修活动有很大帮助 将有助于理解两个关键的DNA修复的内部 小路。

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.