Repair of Clustered DNA Damages

修复DNA簇损伤

• 批准号: 6838686
• 负责人: YOKE W KOW
• 金额: $ 21.66万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-11 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6838686
• 关键词: DNA damage; DNA repair; Escherichia coli; N glycosidase; bacterial genetics; bacterial proteins; endonuclease; enzyme activity; ionizing radiation; microorganism culture; plasmids; radiation genetics

DESCRIPTION (provided by applicant): Ionizing radiation produces a wide spectrum of DNA damages including base and sugar damages, single and double strand breaks, abasic sites, DNA-protein and DNA-DNA crosslinks as well as multiply damage sites(MIDS). Double strand breaks are repaired either by homologous recombination or non-homologous end-joining mechanism. Despite the fact that double strand break is a lethal lesion, lethality induced by ionizing radiation cannot be fully explained by the amount of double strand breaks formed. It was suggested by Ward that a significant amount of cell killing by low LET radiation at biological relevant doses is due to the production of MDS, a cluster of damages within a localized region. There is increasing evidence that MDS are biologically important and might contribute significantly to lethality and mutagenesis induced by ionizing radiation. The long-term goal for this project is to understand the biological consequences of MDS. Two approaches are taken in order to achieve this goal. Aims 1 to 2 are directed to elucidate the in vivo biological consequences of MDS. A yeast shuttle plasmid, pRS413, containing various MDS will be constructed and used to transform E. coli of various repair backgrounds. The lethality conferred by various MDS will be scored by measuring the survival of damaged pRS413 and the mutagenicity of MDS by direct sequencing of the mutant progeny plasmid obtained after transformation. In addition, in vitro processing of these MDS will be studied in Aims 3 and 4. Aim 3 is directed to examine the in vitro processing of DNA containing tandem lesions by E. coli BER enzymes including endonucleases III and VIII, formamidopyrimidine N-glycosylase and 5? AP endonucleases from E.coli. The nature of the reaction products and the kinetics of removal for each of the lesions within the MDS will be elucidated. In addition, a complete in vitro analysis of the possible enzymatic steps involved in the repair of tandem lesions will also be performed. Aim 4 is directed to examine whether E. coli DNA binding protein, such as HU protein can mediate the sequential repair of closely opposed lesion. It is believed that a comprehensive study involving in vivo biological and in vitro enzymatic studies will provide significant insight into understanding the biological consequences of MDS, and thus the genotoxic and mutagenic effect of ionizing radiation.

描述（由申请人提供）：电离辐射产生宽度 DNA损坏的范围，包括基础和糖损伤，单一和双损伤 链断裂，无碱性部位，DNA-蛋白质和DNA-DNA交联以及 乘以伤害位点（中间）。双链断裂是通过修复的 同源重组或非同源最终结合机制。尽管有 双链断裂是致命的病变，通过电离引起的致死性 辐射无法通过双股断裂的量充分解释 形成。沃德建议，大量的细胞杀死 生物学相关剂量下的LET LET辐射是由于MDS的产生， 本地区域内的一大堆损害赔偿。有越来越多的证据 MD在生物学上很重要，并且可能对 电离辐射引起的致死性和诱变。长期目标 该项目是了解MD的生物学后果。二 采取方法是为了实现这一目标。目标1到2针对 阐明MDS的体内生物学后果。酵母穿梭质粒， PRS413，包含各种MD的PRS413将被构造并用于转化E。 各种维修背景的大肠杆菌。各种MD赋予的致命性 通过测量受损PRS413的存活和诱变性来评分 MDS通过直接测序的突变体后代质粒在 转型。此外，将研究这些MD的体外处理 在目标3和4中。目标3用于检查DNA的体外加工 含有大肠杆菌酶的串联病变，包括核酸内切酶III 和viii，甲米酰咪定N-糖基酶和5？ AP来自 e.coli。反应产物的性质和去除动力学的动力学 MD中的每个病变都将阐明。此外，一个完整的 对修复涉及的可能的酶促步骤的体外分析 串联病变也将进行。 AIM 4被指示检查E.是否E.是否 大肠杆菌DNA结合蛋白，例如HU蛋白可以介导顺序修复 紧密对立的病变。据信，一项涉及的全面研究 体内生物学和体外酶学研究将提供重要的 深入了解MD的生物学后果，从而了解 电离辐射的遗传毒性和诱变作用。