Regulations of DNA Alkylation/Deamination Damage Repair

摘要 生成活性氮物质、烷基化剂和脂质过氧化物自由基 内源性和外源性诱导无数的DNA损伤，这被认为影响了 基因组稳定性、细胞活力和导致多种疾病，如癌症和衰老。等 烷基化的、脱氨基的和乙烯基加成物通常通过内源性预防剂修复， 碱基切除修复（BER），当DNA糖基化酶去除受损的 基地其中，一系列结构不同的受损嘌呤被N- 甲基嘌呤DNA-糖基化酶（MPG），存在于从细菌到人的所有物种中。 关于哺乳动物MPG的结构-功能，已有大量的信息 特别是由于我们和其他实验室的努力，这种酶的体内相互作用 这可能深刻地影响其酶活性，体内修复模式（补丁大小等）， 序列特异性在很大程度上仍然未知。MPG与物理相互作用， 受多种因子刺激，包括hHR 23 A/B（核苷酸切除修复蛋白）和 XRCC 1（BER蛋白）。此外，我们的初步结果表明，BRCA 1直接相互作用， 与并刺激MPG的活性，而AP-核酸内切酶，下一个酶在同一个 BER途径结合几种MPG底物损伤而不催化并抑制MPG活性， 并且值得注意的是，不存在于人细胞中的MPG预修复复合物中。然而，MPG缺乏 其N-末端延伸被APE刺激。因此，这些新的初步观察 提供了基础工作，以测试我们的中心假设，即动态蛋白质-蛋白质 相互作用或翻译后修饰可以调节MPG介导的修复， 自发和诱导烷基化、脱氨和过氧化诱导DNA损伤， 对抗基因组不稳定性和癌症。 在我们之前的资助周期中，我们开发了一种非常精确和敏感的质粒， 监测A和Hx修复的体内方法，包括中间修复的复杂分析 步在下一个资金周期，这种修复分析方法与生物化学相结合， 蛋白质组学和哺乳动物遗传学方法（敲除、突变体和siRNA敲低）将 是一个有价值的工具，以确定基因参与不同步骤的MPG特异性BER途径 并阐明在体内的修复机制的<$A和Hx。此外，直接蛋白质-蛋白质 还将使用体外和体内的相互作用以及详细的酶动力学， 了解MPG特异性修复途径对A和Hx的全面机制， 它们代表两种不同类型的DNA损伤剂。 我们的具体目标是：（1）阐明内毒素A和Hx修复的分子机制 细胞通过确定损伤定向修复补丁大小，修复效率取决于 序列背景包括肿瘤抑制基因p53中的突变热点序列;（2） 阐明MPG特异性BER通路识别基底损伤的机制， 分析BRCA 1在体内和体外的A和Hx修复中的作用;以及（3）阐明BRCA 1在体内和体外的A和Hx修复中的作用。 MPG特异性细胞中基底损伤的识别和裂解后的修复机制 BER途径通过使用各种生物化学、蛋白质组学和哺乳动物遗传（敲除， 突变体和siRNA敲低细胞）方法与体内修复测定组合。 我们的长期目标是全面了解MPG的作用和调节， 哺乳动物BER系统的组成部分，用于修复烷基化，脱氨基，脂质过氧化， 表明人体细胞中的DNA损伤。这项研究的信息也将有助于阐明 BER途径中的其他DNA糖基化酶在抗各种诱变和 毒性DNA损伤预防癌症和衰老。此外，这些知识将使我们 最终设计出调节MPG表达的策略， 治疗目的。

Abstract Reactive nitrogen species, alkylating agents and lipid peroxide radicals generated endogenously and exogenously induce a myriad of DNA lesions, which is thought to affect genomic stability, cellular viability and cause multiple diseases such as cancer and aging. Such alkylated, deaminated and etheno adducts are generally repaired via an endogenous preventive pathway, base excision repair (BER), initiated when a DNA glycosylase removes the damaged base. Among these, a series of structurally diverse damaged purines are repaired by N- methylpurine DNA-glycosylase (MPG), present in all species from bacteria to man. Although a significant amount of information is available about the structure-function of mammalian MPG particularly due to efforts from our and other laboratories, the in vivo interactions of this enzyme which may profoundly affect its enzymatic activity, in vivo repair mode (patch size etc.), sequence specificity remains largely unknown. MPG physically interacts with and can be stimulated by various factors including hHR23A/B (a nucleotide excision repair protein) and XRCC1 (a BER protein). Moreover, our preliminary results show that BRCA1 directly interacts with and stimulates MPG's activity, whereas AP-endonuclease, the next enzyme in the same BER pathway binds several MPG substrate lesions without catalysis and inhibit MPG activity, and notably, not present in MPG pre-repair complex in the human cells. However, MPG lacking its N-terminal extension is stimulated by APE. Thus, these novel preliminary observations provide the ground work to test our central hypothesis that the dynamic protein-protein interactions or post-translational modification may modulate the MPG-mediated repair of spontaneous and induced alkylation, deamination and peroxidation-induced DNA damage to combat genomic instability and cancer. In our previous funding cycle we have developed a very precise and sensitive plasmid based in vivo method to monitor repair of ¿A and Hx including intricate analysis of intermediate repair steps. In the next funding cycle, this repair assay method in combination with biochemical, proteomics and mammalian genetic approach (knock-out, mutant and siRNA knock-down) will be a valuable tool to identify genes involved in different steps of MPG-specific BER pathway and elucidate the repair mechanisms of ¿A and Hx in vivo. Furthermore, direct protein-protein interactions in vitro and in vivo and detailed enzyme kinetics will also be used in order to understand a comprehensive mechanism of MPG-specific repair pathway(s) for ¿A and Hx, which are representative of two different classes of DNA damaging agents. Our specific aims are to: (1) elucidate the molecular mechanisms of repair of ¿`A and Hx inside the cells by determining the lesion-directed repair patch size, and repair efficiency depending on sequence context including mutation hotspot sequences in tumor suppressor gene, p53; (2) elucidate the mechanism of recognition of base lesions in MPG-specific BER pathway by analyzing the effect of BRCA1 in ¿A and Hx repair in vivo and in vitro; and (3) elucidate the repair mechanisms subsequent to recognition and cleavage of base lesions in MPG-specific BER pathway by using various biochemical, proteomics, and mammalian genetic (knock-out, mutant and siRNA knock-down cells) approaches in combination with in vivo repair assay. Our long-term goal is comprehensive understanding of the role and regulation of MPG as a component of mammalian BER system for repair of alkylation, deamination, lipid-peroxidation- indiced DNA damage in human cells. The information from this study will also help to elucidate the function of other DNA glycosylases in BER pathway in combating various mutagenic and toxic DNA lesions in preventing cancer and aging. Furthermore, this knowledge will allow us eventually to devise strategies for modulating MPG expression for chemopreventive and therapeutic purposes.