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影响。癌症和发育缺陷与先天性DNA修复缺陷有关。癌症治疗通常基于破坏DNA或抑制病变组织中的DNA修复。在一定程度上，由于其对人类健康的影响，核苷酸切除修复（NER）已经成为近四十年来我们研究的主要焦点和深入研究的主题。很大程度上基于开创性工作在我们的实验室,DNA修复和转录之间的密切关系已经被记载在哺乳动物、酵母和细菌细胞。RNA聚合酶（RNAP）在这一关系中扮演着重要的角色，但我们仍然不清楚它是如何参与的。许多研究结果表明，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矛盾的。这对于人类细胞中的转录偶联修复尤其正确，其中NER和碱基切除修复都涉及。虽然我们的终极目标是理解的机制transcription-coupled尼珥(TC-NER)在人类细胞中,过去的经验的基础上,我们认为重要的一般原则可能揭示了研究过程中最简单的系统,它可以证明。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。即取得证据表明基因的转录T7 RNAP导致增强修复转录链相对于non-transcribed链(TC-NER的标志)紫外光照射后,我们将研究这种效应的生化基础,包括要求其他蛋白质如打和不匹配的蛋白质。2。我们将确定属性的大肠杆菌参与TC-NER RNAP单元测试以及特征突变体(rpoA、rpoB rpoC, rpoD)对紫外线敏感。然后分析紫外线敏感突变体的全球基因组NER和TC-NER。3。我们将测量DNA的销售量在未受损的乳糖操纵子表示,或压抑,比较“无偿”修复合成的频率在每个链使用一个方法在本实验室开发。此外,胸腺嘧啶剥夺后修复合成的性质,其依赖的转录,这可能反映了“无偿”TC-NER的可能性将被评估。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叶酸剥夺对人类的不良后果有关。

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.