STRAND-SPECIFICITY OF DNA REPAIR AND UV INDUCED MUTATION

这项研究的长期目标是了解 核苷酸切除效率中的基因组异质性 哺乳动物的修复与DNA修复调控因素 细胞。目前哺乳动物细胞中DNA修复的模型假定 DNA损伤在转录链上的修复速度比 活跃表达的基因的非转录的链， 优先修复转录链上的损伤是直接 再加上抄写。然而，最近的发现提出了 关于这种模式的普遍性的严重问题。 CPD在该区域的两条DNA链上的快速修复也是 出现在aprt启动子缺失突变体中，其中没有 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，两条链上的CPD都被非常低效地修复。我们的 这些发现似乎挑战了当前的教条，即优先维修 在活跃表达的基因中的DNA损伤 转录偶联修复转录的结果- 阻断病变，并暗示其他机制可能是 在控制哺乳动物核苷酸切除修复中的重要作用 细胞。我们最近又发现了另一个 转录基因正好在aprt基因的下游。我们建议 确定CPD在两条DNA链上的快速修复 在APRT位点上观察到的取决于链特异的， 转录偶联修复多个紧密间隔的、活跃的 在这个区域转录的基因，或者是独立于转录的。 我们假设CPD修复的显著不同 两种不同APRT启动子缺失的观察 突变可能是由于缺失一个 上游顺式作用控制元件通常负责 保持一种“开放”的染色质构型 区域的高效修复，或缺失介导的 导致“位置”的染色体区域并置 显著影响CPD修复效率的“效果” 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，精通和缺陷的DNA修复 内源性aprt基因在CHO细胞中的表达 相对于其原始染色体/转录的“翻转” 定位。这些细胞系将允许评估 链特异的转录偶联修复和 超前/滞后链合成的微分保真度 紫外线诱导的aprt基因座突变的链偏向。

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.