The Role RNA Polymerase II in Transcription Coupled Nucleotide Excision Repair

生物体采用多种保守途径来修复不同类型的DNA损伤。一个重要的途径是核苷酸切除修复（NER），它负责去除大体积的螺旋扭曲DNA损伤，如紫外线诱导的光产物。一种特殊的NER通路，称为转录偶联NER (transcription coupled NER, TC-NER)，是指活跃转录基因的转录链中的优先修复。在酿酒酵母中，人类Cockaynes综合征补体B组蛋白的同源物Rad26和RNA聚合酶II （Pol II）的非必需亚基Rpb9分别介导两种TC-NER机制。Rpb4是Pol II的另一个非必需亚基，在调节TC-NER中起双重作用：抑制Rpb9介导的TC-NER和促进Rad26介导的TC-NER。最近，人们发现第三种TC-NER机制也存在，该机制不依赖于Rad26和Rpb9，但通常被转录延伸/抑制因子Spt4抑制。然而，目前对TC-NER在真核细胞中的分子机制知之甚少。这个项目包含三个目标。目的一是探讨Rad26、Rpb4、Spt4和Pol II之间的相互作用。我们将分析Rad26在调控Spt4的细胞水平以及调节Spt4与Pol II的关联中的作用。我们将研究Rpb4在紫外照射后将Rad26装载到Pol II络合物中的作用。Rpb4和Spt4在与核心Pol II结合过程中的协同作用将被研究。目的二是研究Pol II最大亚基Rpb1的酰化与TC-NER之间的关系。Rpb1在紫外线照射下被小泛素样修饰物（SUMO）共价修饰，并且这种修饰在TC-NER缺陷细胞中增强并持续存在。Rpb1 sumoylation在TC-NER或TC-NER抑制中的作用将通过突变Rpb1上的sumoylation位点来研究。目的三是鉴定与TCR-NER有关的必需Pol II亚基中的关键残基。在Pol II的12个亚基（Rpb1 -12）中，有10个亚基（即Rpb4和Rpb9以外的亚基）是细胞生存所必需的。通过分离和鉴定缺乏TC-NER或“超级精通”TC-NER的Pol II突变体，可以确定这些参与调节TC-NER的基本Pol II亚基中的关键残基。这些研究的完成有望使我们对真核细胞中极其复杂的TC-NER机制的理解达到更高的水平。从拟议的研究中产生的重要发现不仅将发表在科学期刊上，在地方、国家和国际科学会议上提出，而且还将以非科学家公众可以理解的形式进行分析和解释。该项目包括一个重要的教育组成部分，大部分资金用于未来专业人员的研究培训，包括资助实验室成员在科学会议上展示他们的数据。新开发的方法和研究结果将在课堂上展示。此外，本科研究经历将被强调为研究的一个组成部分。

Living organisms employ multiple conserved pathways to repair different types of DNA lesions. One important pathway is nucleotide excision repair (NER), which is responsible for removal of bulky helix-distorting DNA lesions, such as UV induced photoproducts. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of actively transcribed genes. In the yeast Saccharomyces cerevisiae, Rad26, the homologue of the human Cockaynes syndrome complementation group B protein, and Rpb9, a nonessential subunit of RNA polymerase II (Pol II), mediate two TC-NER mechanisms, respectively. Rpb4, another nonessential subunit of Pol II, plays a dual role in regulating TC-NER: suppressing Rpb9 mediated TC-NER and facilitating Rad26 mediated TC-NER. Recently, it was found that a third TC-NER mechanism that is independent of both Rad26 and Rpb9 but is normally suppressed by Spt4, a transcription elongation/suppression factor, also exists. At present, however, little is known about the molecular mechanism of TC-NER in eukaryotic cells. This project contains three objectives. Objective I is to examine interplay among Rad26, Rpb4, Spt4 and Pol II. The roles of Rad26 in regulating the cellular level of Spt4, and in modulating the association of Spt4 with Pol II will be analyzed. The role of Rpb4 in the loading of Rad26 to the Pol II complex following UV irradiation will be examined. The coordination among Rpb4 and Spt4 in their binding to core Pol II will be investigated. Objective II is to investigate the relationship between sumoylation of Rpb1, the largest subunit of Pol II, and TC-NER. Rpb1 is covalently modified by the small ubiquitin-like modifier (SUMO) in response to UV irradiation, and the modification is enhanced and persistent in TC-NER deficient cells. The role of Rpb1 sumoylation in TC-NER or in suppression of TC-NER will be examined by mutating the sumoylation sites on Rpb1. Objective III is to identify critical residues in the essential Pol II subunits that are implicated in TCR-NER. Ten out of twelve subunits (Rpb1 -12) of Pol II, i.e., those subunits other than Rpb4 and Rpb9, are essential for cell viability. The critical residues in these essential Pol II subunits that are involved in modulating TC-NER will be identified by isolating and characterizing Pol II mutants that are deficient in TC-NER or "super-proficient" in TC-NER. Fulfillment of the proposed studies is expected to bring our understanding of the extremely complicated TC-NER mechanism in eukaryotic cells to a significantly higher level. Important findings generated from the proposed studies will not only be published in scientific journals, presented at local, national and international scientific meetings, but also be analyzed and interpreted in formats understandable to the non-scientist public. This project includes a significant educational component, with most of the funding used for research training of future professionals, including funding for lab members to present their data at scientific meetings. New methods developed and findings generated from the studies will be presented in the classroom. In addition, undergraduate research experiences will be emphasized as an integral part of the research.