Regulations of DNA Alkylation/Deamination Damage Repair

DNA烷基化/脱氨基损伤修复的调控

• 批准号: 8658501
• 负责人: RABINDRA ROY
• 金额: $ 6.26万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8658501
• 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 产生的活性氮种，烷基化剂和脂质过氧化物自由基 内源和外源诱导无数的DNA病变，被认为会影响 基因组稳定性，细胞活力并引起多种疾病，例如癌症和衰老。 烷基化，脱水和乙诺代合力通常通过内源预防进行修复 当DNA糖基酶消除损伤时，途径，基本惊喜维修（BER）开始 根据。其中，N-修复了一系列结构上多样化的王子 从细菌到人的所有物种中都存在于甲基嘌呤DNA-糖基化酶（MPG）。虽然 有关哺乳动物MPG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，该酶的体内相互作用 这可能会深刻影响其酶活性，体内修复模式（贴片大小等）， 序列特异性在很大程度上仍然未知。 mpg与物理互动，可以是 受到各种因素的刺激，包括HHR23A/B（核苷酸切除修复蛋白）和 XRCC1（BER蛋白）。此外，我们的初步结果表明BRCA1直接相互作用 使用并刺激MPG的活性，而AP-核酸酶的下一个酶是相同的 BER途径结合了几个MPG底物病变，而无需催化并抑制MPG活性， 值得注意的是，不存在于人类细胞中的MPG预修复复合物中。但是，MPG缺乏 它的N末端延伸由APE刺激。那是这些新颖的初步观察 提供地面工作来测试我们的中心假设，即动态蛋白质蛋白 相互作用或翻译后修改可能会调节MPG介导的修复 赞助和诱导的烷基化，脱氨酸和过氧化诱导的DNA损伤 战斗基因组不稳定性和癌症。 在我们以前的融资周期中，我们开发了一种非常精确和敏感的质粒 监测A和HX修复的体内方法，包括中等修复的复杂分析 步骤。在下一个融资周期中，这种维修测定方法与生化， 蛋白质组学和哺乳动物遗传方法（敲除，突变和siRNA敲除）将 成为识别参与MPG特异性BER途径不同步骤的基因的宝贵工具 并阐明体内A和HX的修复机制。此外，直接蛋白质蛋白质 体外和体内和详细的酶动力学的相互作用也将用于 了解A和HX的MPG特异性修复途径的综合机制， 代表两种不同类别的DNA损伤剂。 我们的具体目的是：（1）阐明修复的分子机制。 通过确定病变定向修复贴片的大小，并根据 序列上下文，包括肿瘤抑制基因中的突变热点序列，p53； （2） 通过通过 分析BRCA1在体内和体外的A和HX修复中的作用； （3）阐明 修复机制是在MPG特异性中识别和裂解碱病变 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敲除细胞）与体内修复测定法结合接近。 我们的长期目标是对MPG的作用和调节的全面理解 哺乳动物BER系统的组成部分，用于修复酒精，死亡，脂质过氧化 - 人类细胞中的指定DNA损伤。这项研究的信息还将有助于阐明 其他DNA糖基酶在BER途径中的功能在对抗各种诱变和 预防癌症和衰老的有毒DNA病变。此外，这些知识将使我们 甚至制定策略来调节MPG表达化学预防和 治疗目的。

项目成果

Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats.

Long-Evans Cinnamon 大鼠自发性肝肿瘤发生过程中肿瘤抑制因子 Tsc2 和 DNA 修复糖基化酶 Nth1 的抑制。

• DOI: 10.1007/s11010-009-0357-1
• 发表时间: 2010
• 期刊: Molecular and cellular biochemistry
• 影响因子: 4.3
• 作者: Sajankila,ShyamaPrasad;Manthena,PraveenV;Adhikari,Sanjay;Choudhury,Sujata;Izumi,Keisuke;Roy,Rabindra
• 通讯作者: Roy,Rabindra

Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase.

• DOI: 10.1016/j.dnarep.2009.06.005
• 发表时间: 2009-10-02
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Adhikari, Sanjay;Uren, Aykut;Roy, Rabindra
• 通讯作者: Roy, Rabindra

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction.

• DOI: 10.1186/1756-0500-5-134
• 发表时间: 2012-03-09
• 期刊: BMC research notes
• 影响因子: 1.8
• 作者: Adhikari S;Karmahapatra SK;Karve TM;Bandyopadhyay S;Woodrick J;Manthena PV;Glasgow E;Byers S;Saha T;Uren A
• 通讯作者: Uren A

Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates.

• DOI: 10.1002/jmr.962
• 发表时间: 2009-11
• 期刊: JOURNAL OF MOLECULAR RECOGNITION
• 影响因子: 2.7
• 作者: Jorgensen, Timothy J.;Chen, Kevin;Chasovskikh, Sergey;Roy, Rabindra;Dritschilo, Anatoly;Uren, Aykut
• 通讯作者: Uren, Aykut

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.

• DOI: 10.1016/j.ab.2011.05.008
• 发表时间: 2011-09-01
• 期刊: Analytical biochemistry
• 影响因子: 2.9
• 作者: Adhikari S;Karmahapatra SK;Elias H;Dhopeshwarkar P;Williams RS;Byers S;Uren A;Roy R
• 通讯作者: Roy R