Regulations of DNA Alkylation/Deamination Damage Repair

DNA烷基化/脱氨损伤修复的调控

• 批准号: 8197229
• 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/8197229
• 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; 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; 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; abstracting; 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

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.

抽象的 产生活性氮、烷化剂和脂质过氧化自由基 内源性和外源性诱发无数 DNA 损伤，这被认为会影响 基因组稳定性、细胞活力并导致癌症和衰老等多种疾病。 烷基化、脱氨基和乙烯加合物通常通过内源性预防性修复 碱基切除修复 (BER) 途径，当 DNA 糖基化酶去除受损的碱基时启动 其中，一系列结构受损的嘌呤被N-修复。 甲基嘌呤 DNA 糖基化酶 (MPG) 存在于从细菌到人类的所有物种中。 关于哺乳动物 MPG 的结构功能有大量信息 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 这可能会深刻影响其酶活性、体内修复模式（贴片大小等）， MPG 与 MPG 的物理相互作用仍然未知。 受到多种因素的刺激，包括 hHR23A/B（一种核苷酸切除修复蛋白）和 此外，我们的初步结果表明，XRCC1（一种 BER 蛋白）与 BRCA1 直接相互作用。 并刺激 MPG 的活性，而 AP-核酸内切酶（同一酶中的下一个酶） BER途径在没有催化的情况下结合多个MPG底物病变并抑制MPG活性， 值得注意的是，人类细胞中的 MPG 预修复复合物中不存在，但 MPG 缺乏。 它的 N 末端延伸受到 APE 的刺激，因此，这些新颖的初步观察结果。 为检验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会调节 MPG 介导的修复 自发和诱导的烷基化、脱氨和过氧化诱导的 DNA 损伤 对抗基因组不稳定性和癌症。 在我们之前的融资周期中，我们开发了一种非常精确和敏感的质粒，基于 体内监测修复的方法 ¿ A 和 Hx 包括中间修复的复杂分析 在下一个资助周期中，这种修复检测方法与生化相结合， 蛋白质组学和哺乳动物遗传学方法（敲除、突变和 siRNA 敲除）将 是识别参与 MPG 特异性 BER 途径不同步骤的基因的有价值的工具 并阐明 ¿ 的修复机制A和Hx体内。 体外和体内的相互作用以及详细的酶动力学也将被用来 了解 MPG 特异性修复途径的综合机制 ¿ A 和 Hx， 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 ¿ 修复的分子机制。 `A 和 Hx 里面 通过确定针对病变的修复补丁大小和修复效率来确定细胞 序列背景，包括肿瘤抑制基因 p53 中的突变热点序列 (2) 阐明 MPG 特异性 BER 通路中基础病变的识别机制 分析 BRCA1 在 ¿ A 和 Hx 体内和体外修复；以及 (3) 阐明 MPG 特异性基底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、 突变体和 siRNA 敲低细胞）方法与体内修复测定相结合。 我们的长期目标是全面了解 MPG 作为一种 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- 这项研究的信息也将有助于阐明人类细胞中的 DNA 损伤。 BER 途径中其他 DNA 糖基化酶在对抗各种诱变和突变方面的功能 此外，这些知识将使我们能够预防有毒的DNA损伤。 最终设计出调节 MPG 表达的策略，用于化学预防和 治疗目的。