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、烷基化剂和脂质过氧化自由基诱导无数的DNA损伤，这些损伤被认为会影响基因组稳定性、细胞活力并导致多种疾病，如癌症和衰老。这些烷基化、脱胺化和乙二加合物通常通过内源性预防性途径修复，即碱基切除修复（BER），当DNA糖基化酶去除受损的碱基时启动。其中，N-甲基嘌呤dna -糖基化酶（MPG）修复了一系列结构多样的受损嘌呤，这些嘌呤存在于从细菌到人类的所有物种中。尽管由于我们和其他实验室的努力，关于哺乳动物MPG的结构功能有了大量的信息，但这种酶在体内的相互作用可能会深刻影响其酶活性，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可与hHR23A/B（一种核苷酸切除修复蛋白）和XRCC1（一种BER蛋白）等多种因子相互作用并受到刺激。此外，我们的初步结果表明，BRCA1直接与MPG的活性相互作用并刺激MPG的活性，而ap -内切酶，同一BER途径中的下一个酶，在没有催化的情况下结合几个MPG底物损伤并抑制MPG的活性，值得注意的是，在人类细胞的MPG预修复复合体中不存在。然而，缺乏n端延伸的MPG受到APE的刺激。因此，这些新颖的初步观察结果为验证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介导的自发和诱导烷基化、脱胺和过氧化诱导的DNA损伤的修复，以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基于质粒的体内方法来监测5A和Hx的修复，包括对中间修复步骤的复杂分析。在下一个资助周期中，这种修复实验方法将结合生化、蛋白质组学和哺乳动物遗传学方法（敲除、突变和siRNA敲除），将成为鉴定mpg特异性BER通路不同步骤参与基因的有价值的工具，并阐明5A和Hx在体内的修复机制。此外，还将使用体外和体内的直接蛋白-蛋白相互作用以及详细的酶动力学来了解作为两种不同类型DNA损伤剂代表的5A和Hx的mpg特异性修复途径的综合机制。我们的具体目标是：(1)通过确定肿瘤抑制基因p53突变热点序列等序列背景下的损伤导向修复斑块大小和修复效率，阐明细胞内5A和Hx修复的分子机制；(2)通过分析BRCA1在体内和体外5A和Hx修复中的作用，阐明mpg特异性BER通路中碱基病变的识别机制；(3)利用各种生化、蛋白质组学和哺乳动物遗传学（敲除、突变和siRNA敲除细胞）方法，结合体内修复实验，阐明mpg特异性BER通路中碱基病变识别和切割后的修复机制。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修复烷基化、脱胺、脂质过氧化诱导的人类细胞DNA损伤中的作用和调控。本研究的信息也将有助于阐明BER通路中其他DNA糖基酶在对抗各种诱变和毒性DNA损伤、预防癌症和衰老中的作用。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：对细胞DNA的损害导致突变和癌症和神经变性的发展。人类细胞中的DNA每天要经历数千到数百万次的破坏事件，这些事件是由包括烟草烟雾在内的环境污染物和内部代谢（内源性）过程产生的。这种DNA加合物通过内源性预防性途径修复，即碱基切除修复（BER）。本项目的目的是了解BER通路的调控机制，并制定调节BER基因表达的策略，以提高化学预防和治疗的疗效。

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.