Regulations of DNA Alkylation/Deamination Damage Repair

摘要 产生的活性氮物种、烷化剂和过氧化脂质 内源性和外源性导致无数的DNA损伤，这被认为是影响 基因组的稳定性，细胞的活力，并导致多种疾病，如癌症和衰老。是这样的 烷基化、脱胺和乙烯加合物通常通过内源性预防性药物修复。 碱基切除修复(BER)途径，当DNA糖基酶去除受损的 基地。其中，一系列结构多样的受损嘌呤被N-修复。 甲基嘌呤DNA糖基酶(MPG)，存在于从细菌到人类的所有物种中。尽管一个 关于哺乳动物MPG的结构和功能有大量的信息可用 特别是由于我们和其他实验室的努力，这种酶在体内的相互作用 这可能会深刻影响其酶活性、体内修复模式(贴片大小等)、 序列特异性在很大程度上仍不清楚。MPG与物理上相互作用，并且可以 受到多种因素的刺激，包括hHR23A/B(核苷酸切除修复蛋白)和 XRCC1(一种BER蛋白)。此外，我们的初步结果表明，BRCA1直接相互作用 与和刺激MPG的活性，而AP-内切酶，同一 BER途径在无催化的情况下结合多个MPG底物损伤，抑制MPG活性， 值得注意的是，在人类细胞的MPG修复前复合体中不存在。然而，MPG缺乏 它的N端延伸是由类人猿刺激的。因此，这些新颖的初步观察结果 为测试我们的中心假设提供了基础工作，即动态蛋白质-蛋白质 相互作用或翻译后修饰可能调节MPG介导的修复 自发性和诱导性烷基化、脱胺和过氧化诱导的DNA损伤 与基因组不稳定和癌症作斗争。 在我们之前的资金周期中，我们开发了一种非常精确和敏感的质粒，基于 监测A和HX修复的活体方法，包括对中间修复的复杂分析 台阶。在下一个资金周期中，这种修复化验方法结合生化， 蛋白质组学和哺乳动物遗传方法(敲除、突变和siRNA敲除)将 是鉴定MPG特异性BER通路不同步骤相关基因的有价值的工具 阐明了A和HX在体内的修复机制。此外，直接蛋白质-蛋白质 还将使用体外和体内的相互作用和详细的酶动力学，以便 了解MPG-A和HX特异性修复途径的全面机制(S)， 它们代表了两种不同类别的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 通过确定细胞的损伤定向修复斑块的大小，以及修复效率取决于 包括抑癌基因p53突变热点序列的序列背景；(2) 阐明MPG特异性BER通路中碱基损伤的识别机制 分析BRCA1在体内和体外修复A和HX中的作用；以及(3)阐明BRCA1在体内和体外的作用 MPG特异性碱基损伤识别和裂解后的修复机制 BER途径通过使用各种生化、蛋白质组学和哺乳动物遗传(敲除， 突变和siRNA敲除细胞)方法与体内修复试验相结合。 我们的长期目标是全面了解MPG作为 哺乳动物BER系统的组成部分，用于修复烷基化、脱氨、脂质过氧化- 被指控对人类细胞造成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糖基酶在对抗多种诱变剂和 有毒DNA损伤在预防癌症和衰老方面的作用。此外，这种知识将使我们能够 最终设计出调控MPG表达的策略，用于化学预防和 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.