Regulations of DNA Alkylation/Deamination Damage Repair

描述(申请人提供)：内源性和外源性产生的活性氮物种、烷化剂和脂质过氧化自由基会导致无数的DNA损伤，被认为会影响基因组的稳定性和细胞的生存能力，并会导致多种疾病，如癌症和衰老。这种烷基化、脱氨基和烯基加合物通常通过一种内源性的预防性途径修复，即碱基切除修复(BER)，当DNA糖基酶去除受损的碱基时，碱基切除修复(BER)启动。其中，一系列结构不同的损伤的嘌呤由N-甲基嘌呤DNA糖基酶(MPG)修复，从细菌到人类的所有物种中都存在这种酶。尽管由于我们和其他实验室的努力，关于哺乳动物MPG的结构和功能已经有了大量的信息，但这种酶在体内的相互作用可能深刻地影响其酶活性，体内修复模式(斑块大小等)，序列特异性在很大程度上仍不清楚。MPG与hHR23A/B(核苷酸切除修复蛋白)和XRCC1(BER蛋白)等多种因素相互作用，并可受其刺激。此外，我们的初步结果表明，BRCA1直接与MPG相互作用并刺激MPG的活性，而同一BER途径中的下一种酶AP-内切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，在人类细胞中不存在MPG预修复复合体中。然而，缺少N-末端延伸的MPG是由APE刺激的。因此，这些新的初步观察为检验我们的中心假设提供了基础工作，该假设认为动态的蛋白质-蛋白质相互作用或翻译后修饰可能调节MPG介导的自发和诱导的烷基化、脱氨和过氧化诱导的DNA损伤的修复，以对抗基因组不稳定性和癌症。在我们之前的资金周期中，我们开发了一种非常精确和敏感的基于在体质粒的方法来监测5A和HX的修复，包括对中间修复步骤的复杂分析。在下一个资金周期，这种修复分析方法与生化、蛋白质组学和哺乳动物遗传学方法(敲除、突变和siRNA敲除)相结合，将是识别MPG特异性BER途径不同步骤所涉及的基因和阐明5A和HX体内修复机制的有价值的工具。此外，还将利用体外和体内直接的蛋白质-蛋白质相互作用和详细的酶动力学来了解代表两类不同DNA损伤剂的MPG特异性修复途径(S)的全面机制。我们的具体目标是：(1)通过测定5A和HX在细胞内的修复斑块大小，阐明细胞内5A和HX修复的分子机制，以及修复效率取决于序列背景，包括抑癌基因P53中的突变热点序列；(2)通过分析BRCA1在5A和HX修复体内和体外的作用，阐明MPG特异性BER途径中碱基损伤的识别机制；利用多种生化、蛋白质组学和哺乳动物遗传学方法(敲除细胞、突变细胞和siRNA敲除细胞)，结合体内修复实验，阐明MPG特异性BER途径中碱基损伤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系统的一部分在修复烷基化、脱氨和脂质过氧化导致的人类细胞DNA损伤中的作用和调节。这项研究的信息也将有助于阐明BER途径中的其他DNA糖基酶在对抗各种突变和毒性DNA损伤预防癌症和衰老方面的功能。此外，这一知识将使我们最终能够设计出调控MPG表达的策略，用于化学预防和治疗目的。与公共卫生相关：细胞DNA的损伤会导致癌症和神经变性的突变和发展。人体细胞中的DNA每天经历几千到几百万次破坏事件，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。这种DNA加合物通过一种内源性的预防性途径--碱基切除修复(BER)来修复。本项目的目的是了解BER途径的调节机制，并设计调控BER基因表达的策略，以提高化疗药物和治疗药物的疗效。

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.