Regulations of DNA Alkylation/Deamination Damage Repair

描述（由申请人提供）：内源性和外源性产生的活性氮物质、烷化剂和脂质过氧化物自由基诱导无数DNA损伤，认为其影响基因组稳定性、细胞活力并引起多种疾病，如癌症和衰老。这种烷基化、脱氨基和乙烯基加合物通常通过内源性预防途径修复，即碱基切除修复（BER），当DNA糖基化酶去除受损碱基时启动。其中，一系列结构上不同的受损嘌呤被N-甲基嘌呤DNA-糖基化酶（MPG）修复，该酶存在于从细菌到人的所有物种中。尽管特别是由于我们和其他实验室的努力，可以获得关于哺乳动物MPG的结构-功能的大量信息，但是这种酶的体内相互作用可能深刻地影响其酶活性，体内修复模式（贴片尺寸等），序列特异性在很大程度上仍然未知。MPG与各种因子物理相互作用并可被各种因子刺激，所述因子包括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，并刺激MPG的活性，而AP-内切核酸酶，在同一BER途径中的下一个酶结合几个MPG底物病变没有催化和抑制MPG活性，值得注意的是，不存在于MPG修复前复合物在人类细胞。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介导的自发和诱导的烷基化，脱氨基和过氧化诱导的DNA损伤的修复，以打击基因组不稳定性和癌症。在我们之前的资助周期中，我们已经开发了一种非常精确和灵敏的基于质粒的体内方法来监测5A和Hx的修复，包括中间修复步骤的复杂分析。在下一个资助周期中，这种修复检测方法与生物化学、蛋白质组学和哺乳动物遗传学方法（敲除、突变和siRNA敲除）相结合，将是一种有价值的工具，用于鉴定参与MPG特异性BER途径不同步骤的基因，并阐明5A和Hx的体内修复机制。此外，还将使用体外和体内的直接蛋白质-蛋白质相互作用和详细的酶动力学，以了解5A和Hx的MPG特异性修复途径的综合机制，这两种不同类型的DNA损伤剂的代表。我们的具体目标是：（1）通过确定损伤定向修复斑块大小和修复效率（取决于包括肿瘤抑制基因p53中的突变热点序列的序列背景）来阐明细胞内5A和Hx修复的分子机制：（2）通过分析BRCA 1在体内和体外5A和Hx修复中的作用来阐明MPG特异性BER途径中碱基损伤的识别机制;和（3）通过使用各种生物化学、蛋白质组学和哺乳动物遗传学（敲除、突变体和siRNA敲低细胞）方法结合体内修复测定来阐明MPG特异性BER途径中碱基损伤的识别和切割之后的修复机制。我们的长期目标是全面了解MPG作为哺乳动物BER系统的一个组成部分在人类细胞中修复烷基化，脱氨基，脂质过氧化诱导的DNA损伤中的作用和调控。本研究的信息也将有助于阐明BER途径中其他DNA糖基化酶在对抗各种致突变和毒性DNA损伤以预防癌症和衰老中的功能。此外，这些知识将使我们最终能够设计出用于化学预防和治疗目的的调节MPG表达的策略。公共卫生相关性：细胞DNA的损伤导致突变和癌症和神经退行性疾病的发展。人类细胞中的DNA每天经历数千到数百万次破坏性事件，这些事件由环境污染物（包括烟草烟雾）和内部代谢（内源性）过程产生。这样的DNA加合物通过内源性预防途径，碱基切除修复（BER）修复。本项目的目标是了解BER途径的调控机制，并设计调控BER基因表达的策略，以提高化学预防和治疗的功效。

DESCRIPTION (provided by applicant): 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 5A 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 5A 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 5A 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 5A 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 5A 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-induced 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. PUBLIC HEALTH RELEVANCE: Damage to cellular DNA causes mutations and development of cancer and neurodegeneration. The DNA in human cell undergoes several thousand to million damaging events per day, generated by both environmental pollutants including tobacco smoke and internal metabolic (endogenous) processes. Such DNA adducts are repaired by an endogenous preventive pathway, Base Excision Repair (BER). The goal of this project is to understand the mechanisms of regulation of BER pathway and devise strategies for modulating the expression of BER genes to improve the efficacy of chemopreventives and therapeutics.