Transcription coupled DNA repair in E. Coli

DNA损伤和DNA修复对人类健康有许多重要影响。 癌症和发育缺陷与DNA修复的先天性缺陷有关。癌症治疗通常基于损伤DNA或抑制患病组织中的DNA修复。 部分由于其对人类健康的影响，核苷酸切除修复（NER）一直是近四十年来深入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，DNA修复和转录之间的密切关系已经在哺乳动物，酵母和细菌细胞中被证明。 RNA聚合酶（RNAP）是在这种关系中发挥重要作用的主要候选者，但我们仍然不清楚它是如何参与的。 大量的研究结果表明，RNAP与其他参与DNA修复的蛋白质相互作用，但目前关于相互作用细节的想法有时是矛盾的。 这对于其中NER和碱基切除修复两者都涉及的人类细胞中的转录偶联修复尤其如此。虽然我们的最终目标是了解人类细胞中转录偶联NER（TC-NER）的机制，但根据过去的经验，我们认为，通过研究最简单系统中的过程，可以揭示重要的一般原理。 因此，我们将集中在噬菌体T7的单体RNAP和大肠杆菌的多亚基RNAP。I.已经获得的证据表明，转录的基因由T7 RNAP结果在增强修复的转录链相对于非转录链（的标志TC-NER）后，紫外线照射，我们将研究这种效果的生化基础，包括对其他蛋白质，如Mfd和错配蛋白的要求。二.我们将确定E. coli RNAP亚基参与的TC-NER通过测试充分表征的突变体（rpoA，rpoB，rpoC，rpoD）的UV敏感性。 然后将分析UV敏感突变体的全局基因组NER和TC-NER。三. 我们将测量未受损的乳糖操纵子表达或抑制时的DNA周转率，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，修复合成胸腺嘧啶剥夺后，其依赖于转录的性质，以及它可能反映“无偿”TC-NER的可能性将进行评估。 胸腺嘧啶剥夺期间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人类缺乏叶酸的不良后果有关。

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.