STRAND-SPECIFICITY OF DNA REPAIR AND UV INDUCED MUTATION

DNA 修复和紫外线诱导突变的链特异性

• 批准号: 6019305
• 负责人: GERALD M ADAIR
• 金额: $ 14.65万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019305
• 关键词: CHO cells; DNA damage; DNA repair; adenine phosphoribosyltransferase; carcinogen testing; mutagen testing; mutagens; neoplasm /cancer genetics; radiation carcinogen; radiation carcinogenesis; radiation genetics; ultraviolet radiation

The long term goal of this research is to understand the basis for genomic heterogeneity in the efficiency of nucleotide excision repair and the factors that regulate DNA repair in mammalian cells. Current models of DNA repair in mammalian cells presume that repair of DNA damage is faster on the transcribed strand than the nontranscribed strand of actively expressed genes, and that preferential repair of damage on the transcribed strand is directly coupled to transcription. However, recent findings have raised serious questions concerning the universality of such models. Rapid repair of CPD on both DNA strands of this region is also seen in an APRT promoter-deletion mutant in which there is no transcription of APRT gene sequences. However, in a second APRT promoter-deletion mutant with a much larger 5'-extending deletion, CPD on both strands are very inefficiently repaired. Our findings appear to challenge current dogma that preferential repair of DNA damage in actively expressed genes in simply a consequence of transcription-coupled repair of transcription- blocking lesions, and suggest that other mechanisms may be important in controlling nucleotide excision repair in mammalian cells. We have recently discovered another, convergently transcribed gene just downstream of APRT gene. We propose to determine whether the rapid repair of CPD on both DNA strands observed at the APRT locus is dependent upon the strand-specific, transcription-coupled repair of multiple closely-spaced, actively- transcribed genes in this region, or is independent of transcription. We hypothesize that the striking differences in CPD repair observed between our two different APRT promoter-deletion mutants might be a consequence of either the deletion of an upstream cis-acting control element normally responsible for maintaining an "open" chromatin configuration that facilitates efficient repair of the region, or the deletion-mediated juxtaposition of chromosomal regions resulting in "position effects" that can dramatically affect the efficiency of CPD repair over a large genomic domain. Finally, we propose to generate, by targeted gene replacement, DNA repair-proficient and -deficient CHO cell lines in which the endogenous APRT gene has been "flipped" with respect to its original chromosomal/transcriptional orientation. These cell lines will allow assessment of the contributions of strand-specific transcription-coupled repair and differential fidelity of leading/lagging strand synthesis to the strand-bias of UV-induced mutation at the APRT locus.

这项研究的长期目标是了解 核苷酸切除效率的基因组异质性 哺乳动物DNA修复及调节DNA修复的因素 细胞。目前哺乳动物细胞 DNA 修复模型推测 转录链上 DNA 损伤的修复速度比 活跃表达基因的非转录链，并且 直接优先修复转录链上的损伤 与转录耦合。 然而，最近的调查结果提出 关于此类模型的普遍性的严重问题。 CPD 对该区域两条 DNA 链的快速修复也 见于 APRT 启动子缺失突变体，其中没有 APRT基因序列的转录。 然而，下一秒 具有更大 5' 延伸的 APRT 启动子缺失突变体 缺失后，两条链上的 CPD 修复效率非常低。 我们的 研究结果似乎挑战了当前优先修复的教条 简单地分析活跃表达基因中的 DNA 损伤 转录偶联修复的结果- 阻断病变，并表明可能有其他机制 对控制哺乳动物的核苷酸切除修复很重要 细胞。 我们最近发现了另一个收敛的 转录基因位于 APRT 基因下游。 我们建议 确定两条 DNA 链上的 CPD 是否快速修复 在 APRT 位点观察到的结果取决于链特异性， 多个紧密间隔的、主动的转录耦合修复 转录基因在该区域，或者独立于转录。 我们假设 CPD 修复的显着差异 在我们的两个不同的 APRT 启动子缺失之间观察到 突变体可能是缺失一个的结果 上游顺式作用控制元件通常负责 维持“开放”的染色质构型，有利于 该区域的有效修复，或删除介导的 染色体区域的并列导致“位置 效应”可以显着影响 CPD 修复的效率 在一个大的基因组域上。最后，我们建议通过以下方式生成 靶向基因替换、DNA 修复熟练和缺乏 内源性 APRT 基因已被导入的 CHO 细胞系 相对于其原始染色体/转录“翻转” 方向。 这些细胞系将允许评估 链特异性转录偶联修复的贡献和 前导/滞后链合成的差异保真度 APRT 基因座紫外线诱导突变的链偏向。