Transcription coupled DNA repair in E. Coli

DNA损伤和DNA修复对人类健康有许多重要的后果。癌症和发育缺陷与DNA修复的先天缺陷有关。癌症的治疗通常是基于破坏病变组织中的DNA或抑制DNA修复。核苷酸切除修复(NER)由于其对人类健康的影响，近40年来一直是密集研究的主题和我们研究的主要焦点。在很大程度上基于我们实验室的开创性工作，已经在哺乳动物、酵母和细菌细胞中证明了DNA修复和转录之间的密切关系。RNA聚合酶(RNAP)是在这一关系中发挥重要作用的最佳候选者，但我们仍然不清楚它是如何参与的。大量研究结果表明，RNAP与参与DNA修复的其他蛋白质相互作用，但目前关于相互作用细节的想法有时是相互矛盾的。这对于人类细胞中的转录偶联修复尤其如此，其中NER和碱基切除修复都被涉及。虽然我们的最终目标是了解转录偶联NER(TC-NER)在人类细胞中的机制，但根据过去的经验，我们相信通过在最简单的系统中研究这一过程可以揭示重要的一般原理。因此，我们将重点研究噬菌体T7的单体RNAP和大肠杆菌的多亚基RNAP。I.有证据表明，在紫外线照射后，T7 RNAP转录导致转录的链相对于非转录的链(TC-NER的标志)的修复增强，我们将研究这种效应的生化基础，包括对其他蛋白质的要求，如MFD和错配蛋白质。我们将通过测试特征良好的突变体(rpoA、rpoB、rpoC、rpoD)来鉴定参与TC-NER的大肠杆菌RNAP亚基的性质。然后对紫外线敏感突变体进行全球基因组NER和TC-NER的分析。我们将测量未受损的乳胶操纵子在表达或抑制时的DNA周转率，并使用本实验室开发的方法比较每条链中“无偿”修复合成的频率。此外，胸腺嘧啶剥夺后的修复合成的性质，它对转录的依赖性，以及它可能反映“无端的”TC-NER的可能性将被评估。胸腺嘧啶剥夺过程中的基因表达谱将通过微阵列进行评估。这些实验的结果与理解叶酸缺乏对人类的不利后果有关。

DNA damage and DNA repair have many important consequences for human health. Cancer and developmental defects have been associated with congenital deficiencies in DNA repair. Cancer treatment is often based upon damaging DNA or inhibiting DNA repair in the diseased tissue. In part because of its implications for human health, nucleotide excision repair (NER) has been the subject of intense investigation and a major focus of our research for nearly four decades. Based largely upon pioneering work in our laboratory, a close relationship between DNA repair and transcription has been documented in mammalian, yeast, and bacterial cells. RNA polymerase (RNAP) is a prime candidate for an essential role in this relationship, but we still do not understand exactly how it participates. Results of numerous investigations indicate that RNAP interacts with other proteins involved in DNA repair, but current ideas about the details of the interactions are sometimes contradictory. This is particularly true for transcription coupled repair in human cells in which both NER and base excision repair have been implicated. Although our ultimate goal is to understand the mechanism of transcription-coupled NER (TC-NER) in human cells, on the basis of past experience we believe that important general principles may be revealed by studying the process in the simplest systems in which it can be demonstrated. Therefore, we will focus upon the monomeric RNAP of bacteriophage T7 and the multisubunit RNAP of Escherichia coli. I. Having obtained evidence that transcription of a gene by the T7 RNAP results in enhanced repair of the transcribed strand relative to the non-transcribed strand (the hallmark of TC-NER) after UV-irradiation, we will study the biochemical basis of this effect, including the requirements for other proteins such as Mfd and mismatch proteins. II. We will identify properties of the E. coli RNAP subunits involved in TC-NER by testing well characterized mutants (rpoA, rpoB, rpoC, rpoD) for UV sensitivity. UV sensitive mutants will then be analyzed for global genomic NER and TC-NER. III. We will measure DNA turnover in the undamaged lac operon when it is expressed, or repressed, comparing the frequency of "gratuitous" repair synthesis in each strand using an approach developed in this laboratory. In addition, the nature of repair synthesis following thymine deprivation, its dependence upon transcription, and the possibility that it may reflect "gratuitous" TC-NER will be assessed. Gene expression profiles during thymine deprivation will be assessed by microarrays. The results of these experiments are relevant to an understanding of the adverse consequences of folate deprivation in humans.