STRAND-SPECIFICITY OF DNA REPAIR AND UV INDUCED MUTATION

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修复及其调控因子 细胞目前哺乳动物细胞中的DNA修复模型推测 DNA损伤的修复在转录链上要比 活跃表达基因的非转录链， 转录链上损伤的优先修复直接 再加上转录。 然而，最近的调查结果表明， 这些模型的普遍性存在严重问题。 该区域的两条DNA链上CPD的快速修复也是 在APRT启动子缺失突变体中观察到， APRT基因序列的转录。 然而，在一秒钟内， APRT启动子缺失突变体具有更大的5 '-延伸 缺失时，两条链上的CPD都非常低效地修复。 我们 这些发现似乎挑战了当前的教条，即优先修复 的DNA损伤在活跃表达的基因中， 转录偶联修复的结果 阻断病变，并表明其他机制可能是 在哺乳动物中控制核苷酸切除修复中重要 细胞 我们最近发现了另一个， 转录基因的下游。 我们建议 确定两条DNA链上CPD的快速修复 在APRT基因座上观察到的依赖于链特异性， 转录偶联修复多个紧密间隔的，积极的， 在该区域中转录基因，或者独立于转录。 我们假设CPD修复的显著差异 在我们的两个不同的APRT启动子缺失之间观察到 突变体可能是一个缺失的结果， 上游顺式作用控制元件通常负责 保持“开放”染色质构型， 该区域的有效修复，或缺失介导的 染色体区域的并置导致“位置 影响”，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 靶向基因置换，DNA修复熟练和缺陷 CHO细胞系，其中内源性APRT基因已被 “翻转”相对于其原始染色体/转录 导向 这些细胞系将允许评估 链特异性转录偶联修复的贡献， 前导链/滞后链合成对 在APRT基因座处UV诱导突变的链偏倚。

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.