REPAIR OF OXIDATIVELY DAMAGED GUANINES IN HUMAN

人体氧化损伤鸟嘌呤的修复

• 批准号: 2896556
• 负责人: A-Lien L Lu-Chang
• 金额: $ 20.99万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-14 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2896556
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; X ray; alkylating agents; aphidicolin; carcinogen testing; chemical carcinogen; chemical carcinogenesis; endonuclease; enzyme activity; guanine; human genetic material tag; human tissue; mutagen testing; mutagens; neoplasm /cancer genetics; nucleic acid sequence; oligonucleotides; oxidative stress; proliferating cell nuclear antigen; tissue /cell culture; ultraviolet radiation

Mismatch repair is an error avoidance pathway devoted to enhancing the fidelity of DNA replication and maintaining genetic stability. Mutations in human mismatch repair genes predispose individuals to cancer. Oxidative stress and metabolic processes which produce reactive oxygen species have been implicated as important causative agents of mutagenesis, carcinogenesis, aging, and a number of diseases. The human MutY homolog (hMYH) mismatch repair pathway for repairing A/G, A/C, and A/8-oxoG mismatches will be our major focus. The 8-oxoG lesion is a major stable product of DNA oxidative damage and has the most deleterious effects because it can mispair with adenine. Thus, A/8-oxoG mismatches are particularly important biological substrates for hMYH. hMYH, like the E. coli MutY protein, is an adenine DNA glycosylase. Because recombinant hMYH protein expressed in E. coli and native hMYH have different mismatch specificities, the structural and functional differences of these proteins will be further analyzed. Glycosylase and apurinic/apyrimidinic (AP) lyase activities on DNA containing A/G, A/C, A/8-oxoG, and other base analogs will be assayed. Our results indicate the hMYH, MutS homologs (hMSH2 and hMSH6), and MutL homologs (hMLH1 and hPMS2) are associated with the DNA replication complex, thus the interactions between hMYH and replicative as well as mismatch repair proteins will be investigated by co-immunoprecipretation and affinity chromatography. The coupling of hMYH repair with DNA replication may direct MYH repair to the misincorporated adenines on the daughter strands. The effect of proliferating cell nuclear antigen (PCNA, an accessory factor for DNA polymerases delta and epsilon) on hMYH activity will be determined. Human breast and lung cancer cells will be analyzed for hMYH expression and screened for mutations in the hMYH gene. The sensitivities of the MYH defective cells to oxidative agents and radiation will be analyzed. Through the study of the mechanism of DNA mismatch repair, our understanding of cancer, aging, and genetic diseases can be advanced.

错配修复是一种错误避免途径，致力于增强 DNA复制的保真度并保持遗传稳定性。 人类错配修复基因的突变使个体容易 癌症。 氧化应激和代谢过程产生反应性 氧物种被认为是重要的致病因素 突变、致癌、衰老和许多疾病。 人类 MutY 同源物 (hMYH) 错配修复途径用于修复 A/G、A/C 和 A/8-oxoG 错配将是我们的主要关注点。 8-oxoG 损伤是 DNA氧化损伤的主要稳定产物，具有最 有害影响，因为它可能与腺嘌呤错配。 因此，A/8-oxoG 错配是 hMYH 特别重要的生物底物。 hMYH 与大肠杆菌 MutY 蛋白一样，是一种腺嘌呤 DNA 糖基化酶。 因为重组hMYH蛋白在大肠杆菌和天然hMYH中表达 具有不同的错配特性、结构和功能 将进一步分析这些蛋白质的差异。 糖基化酶和 无嘌呤/无嘧啶 (AP) 裂解酶对含有 A/G、A/C、 A/8-oxoG 和其他碱基类似物将被测定。 我们的结果表明 hMYH、MutS 同源物（hMSH2 和 hMSH6）和 MutL 同源物（hMLH1 和 hMLH1） hPMS2) 与 DNA 复制复合体相关，因此 hMYH 和复制以及错配修复之间的相互作用 将通过免疫共沉淀和亲和力研究蛋白质 色谱法。 hMYH 修复与 DNA 复制的耦合可能 直接 MYH 修复女儿体上错误掺入的腺嘌呤 股。 增殖细胞核抗原（PCNA，一种 DNA 聚合酶 delta 和 epsilon 的辅助因子）对 hMYH 活性的影响 将被确定。 将分析人类乳腺癌和肺癌细胞 检测 hMYH 表达并筛选 hMYH 基因突变。这 MYH 缺陷细胞对氧化剂的敏感性 将分析辐射。 通过对DNA机制的研究 错配修复，我们对癌症、衰老和遗传的理解 疾病可能会进展。