Processing Site-Specific DNA Lesions by DNA Repair/Recom

通过 DNA 修复/重组处理位点特异性 DNA 损伤

• 批准号: 6990365
• 负责人: Karen M Vasquez
• 金额: $ 16.16万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-21 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990365
• 关键词: CHO cells; DNA damage; DNA repair; cell free system; crosslink; genetic recombination; mitomycin C; psoralens; triple helix; ultraviolet radiation

DNA interstrand crosslinks (ICLs) present a formidable block to DNA metabolic processes and must be repaired for cell survival. While much work has been done to define the mechanism(s) of DNA interstrand crosslink repair in bacteria and yeast, very little is known about their repair in mammalian cells. It is has been proposed that both homologous recombination (HR) and nucleotide excision repair pathways are involved in DNA crosslink repair. It is becoming increasingly evident that there is overlap among the DNA repair pathways for certain types of DNA lesions. Our working hypothesis is that a number of proteins already defined as essential in DNA repair mechanisms are also required for efficient HR, or work together with HR in DNA interstrand crosslink processing and removal. The long-term objectives of the proposed research are to elucidate the molecular mechanism(s) involved in the removal of site-specific DNA interstrand crosslinks and complex DNA lesions (psoralen plus UVA radiation and mitomycin C crosslinks directed by triplex formation) from the mammalian genome, to identify interactions among the DNA repair pathways, and to determine the role(s) of homologous recombination, nucleotide excision, and mismatch repair mechanisms in the removal and processing of ICLs and complex DNA lesions. Specifically we propose to: 1) direct sitespecific DNA crosslinks to the genome in normal and recombination-deficient Chinese hamster ovary cells to elucidate the role of recombination in the removal of interstrand crosslinks and complex DNA lesions; 2) determine the mechanism(s) of removal of site-specific DNA interstrand crosslinks and complex DNA lesions directed by triplex-forming oligonucleotides in mammalian cell-free extracts; and 3) identify interactions between proteins involved in DNA repair pathways in the removal of site-specific DNA interstrand crosslinks and complex DNA lesions.

DNA 链间交联 (ICL) 对 DNA 代谢过程构成强大阻碍，必须进行修复才能保证细胞生存。虽然已经做了很多工作来确定细菌和酵母中 DNA 链间交联修复的机制，但人们对它们在哺乳动物细胞中的修复知之甚少。有人提出，同源重组（HR）和核苷酸切除修复途径都参与DNA交联修复。越来越明显的是，某些类型的 DNA 损伤的 DNA 修复途径之间存在重叠。我们的工作假设是，许多已经被定义为 DNA 修复机制必需的蛋白质也是高效 HR 所必需的，或者与 HR 一起工作 DNA 链间交联处理和去除。拟议研究的长期目标是阐明从哺乳动物基因组中去除位点特异性 DNA 链间交联和复杂 DNA 损伤（补骨脂素加 UVA 辐射和由三链体形成引导的丝裂霉素 C 交联）所涉及的分子机制，以确定 DNA 修复途径之间的相互作用，并确定同源重组的作用， ICL 和复杂 DNA 损伤去除和处理中的核苷酸切除和错配修复机制。具体来说，我们建议：1）直接针对特定地点 DNA 与正常和重组缺陷的中国仓鼠卵巢细胞中基因组的交联，以阐明重组在消除链间交联和复杂 DNA 损伤中的作用； 2) 确定哺乳动物无细胞提取物中由三链体形成寡核苷酸引导的去除位点特异性 DNA 链间交联和复杂 DNA 损伤的机制； 3) 确定参与 DNA 修复途径的蛋白质之间的相互作用，以消除位点特异性 DNA 链间交联和复杂的 DNA 损伤。