Regulations of DNA Alkylation/Deamination Damage Repair

DNA烷基化/脱氨损伤修复的调控

• 批准号: 7996576
• 负责人: RABINDRA ROY
• 金额: $ 25.88万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996576
• 关键词: 3-methyladenine; Affect; Aging; Alkylating Agents; Alkylation; Animals; BRCA1 gene; Back; Bacteria; Base Excision Repairs; Binding; Biochemical; Biological Assay; Cancerous; Carcinogen exposure; Carcinogens; Catalysis; Cells; Chemopreventive Agent; Chromosome abnormality; Complex; DNA; DNA Adducts; DNA Alkylation; DNA Damage; DNA Repair; DNA glycosylase; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; DNA-3-methyladenine glycosidase II; Deamination; Development; Diabetes Mellitus; Disease; Embryo; Environmental Pollutants; Enzyme Interaction; Enzyme Kinetics; Enzymes; Escherichia coli; Eukaryota; Event; Excision; Excision Repair; Fibroblasts; Funding; Genes; Genome Stability; Genomic Instability; Goals; Health; Homologous Gene; Human; Hypoxanthines; In Vitro; Incidence; Knock-out; Knockout Mice; Knowledge; Laboratories; Lesion; Ligation; Lipid Peroxidation; Lipid Peroxides; Lipids; Liver; Malignant Neoplasms; Mammalian Genetics; Mediating; Metabolic; Metabolism; Methods; Methylpurine DNA Glycosylase; Microscopy; Mismatch Repair; Molecular; Monitor; Mus; Mutagenesis; Mutation; N-terminal; Nerve Degeneration; Nuclear; Nucleotide Excision Repair; Nucleotides; Ovarian Tissue; Pathway interactions; Plasmids; Post-Translational Protein Processing; Predisposition; Preventive; Process; Proteins; Proteomics; Purines; Reactive Nitrogen Species; Regulation; Repair Complex; Research; Role; Series; Side; Sister Chromatid Exchange; Small Interfering RNA; Specificity; Structure; System; Testing; Therapeutic; Tissues; Tobacco smoke; Toxic Environmental Substances; Toxic effect; Tumor Suppressor Genes; Urethane; Vinyl Chloride; Work; XRCC1 gene; adduct; base; carcinogenesis; combat; human XRCC1 protein; improved; in vivo; knock-down; man; mutant; novel; peroxidation; polymerization; prevent; protein protein interaction; purine; reconstitution; repaired; tool

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.

描述（由申请人提供）：反应性氮，烷基化剂和脂质过氧化物自由基内源和外源会诱导无数的DNA病变，这被认为会影响基因组稳定性，细胞生存能力并引起多种疾病，例如癌症和衰老。当DNA糖基化酶去除受损的碱时，这种烷基化，脱氧和乙烯加合物通常通过内源预防途径，碱基切除修复（BER）修复。其中，通过细菌到人的所有物种，都存在一系列结构上多样化的嘌呤，通过N-甲基嘌呤DNA-糖基化酶（MPG）修复。尽管有关哺乳动物MPG的结构功能的大量信息，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可能会深刻影响其酶活性，在体内修复模式（贴片大小等），但序列特异性仍然很大。 MPG与各种因素（包括HHR23A/B（A核苷酸切除修复蛋白）和XRCC1（BER蛋白）（BER蛋白））进行物理相互作用并可以刺激。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了MPG的活性，而AP-核酸酶，同一BER途径中的下一个酶结合了几种MPG底物病变，而无需催化和抑制MPG活性，并且不存在于人类细胞中的MPG Prepair Confffect中。然而，猿刺激了缺乏其N末端扩展的MPG。因此，这些新颖的初步观察结果提供了基础工作，以测试我们的中心假设，即动态蛋白 - 蛋白质相互作用或翻译后修饰可能调节MPG介导的自发性和诱导烷基化，脱氨酸以及过氧化诱导的DNA损害的DNA损害对基因组不稳定和癌症的损害。在我们以前的融资周期中，我们开发了一种非常精确且敏感的基于体内方法，以监测5A和HX的修复，包括对中间修复步骤的复杂分析。在下一个融资周期中，这种修复测定方法与生化，蛋白质组学和哺乳动物遗传方法（敲除，突变体和siRNA敲除）结合使用，将是一种有价值的工具，可以识别涉及MPG特异性BER途径不同步骤的基因，并阐明5A和HX InVivo的修复机制。此外，还将使用直接的蛋白质 - 蛋白质相互作用，体内和体内和详细的酶动力学，以了解5A和HX的MPG特异性修复途径的综合机制，这些机制代表了两种不同类别的DNA损害剂。我们的具体目的是：（1）通过确定病变定向的修复贴片大小，阐明细胞内5A和HX修复的分子机制，并根据序列上下文的维修效率，具体取决于序列上下文，包括包括肿瘤抑制基因中的突变热点序列，p53； （2）通过分析BRCA1在5A中的效果和体内和体外的HX修复的作用，阐明了MPG特异性BER途径中基本病变的识别机理； （3）通过使用各种生化，蛋白质组学和哺乳动物遗传学（敲除，突变和siRNA敲除型细胞）与体内修复测定法相结合的方法，阐明了MPG特异性BER途径中碱病变识别和切割的修复机制。我们的长期目标是对MPG的作用和调节的全面了解，作为修复烷基化，脱氨酸，脂质过氧化诱导的DNA损伤的哺乳动物BER系统的组成部分。这项研究的信息还将有助于阐明其他DNA糖基化酶在BER途径中在防止癌症和衰老中打击各种诱变和有毒DNA病变的功能。此外，这些知识将使我们最终制定策略，以调节MPG表达以进行化学预防和治疗目的。公共卫生相关性：细胞DNA的损害会导致突变以及癌症和神经退行性的发展。人类细胞中的DNA每天经历数千至百万的破坏性事件，这是由烟草烟雾和内源性（内源性）过程在内的两种环境污染物产生的。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。该项目的目的是了解BER途径调节的机制，并制定了调节BER基因表达以提高化学预防和治疗剂的功效的策略。