Transcription Coupled DNA Repair in S. cerevisiae

描述（由申请人提供）：生物体采用多种保守途径来修复不同类型的DNA损伤。其中一个重要的途径是转录偶联修复（TCR），它负责活性基因的转录链的快速修复。TCR缺陷会导致严重的人类疾病柯凯因综合症，以及其他可能尚未确定的疾病。Rpb9是RNA聚合酶II （Pol II）的一个非必需亚基，最近被发现介导了一个TCR亚通路，该亚通路在Pol II转录基因中叠加在Rad26介导的TCR亚通路上。Rpb4是Pol II的另一个非必需亚基，可能在调节两种TCR亚通路中发挥双重作用，抑制Rpb9介导的TCR，促进Rad26介导的TCR。Rpb9介导TCR所需的结构域和关键残基将通过系统地从N端和c端删除残基，并通过替换某些关键残基来绘制。其他参与Rpb9介导的TCR的蛋白将被标记为Rpb9或Rad14的表位拉下。提取的蛋白质将通过质谱分析进行鉴定，并对其进行遗传和生物化学表征。酵母双杂交系统也可用于筛选参与Rpb9介导的TCR的潜在蛋白。Pol II最大亚基的共价修饰响应DNA损伤可能作为TCR信号。在一定的TCR条件下，Defl参与了Pol II的泛素化和降解。Rpb9和Defl在Pol II最大亚基共价修饰中的作用将通过Western blot分析来检验。抑制Rpb9介导的TCR所需的Rpb4结构域将通过系统地删除蛋白质不同区域的残基来绘制。Rpb7是Pol II的重要亚基，可与Rpb4形成络合物。Rpb7在抑制Rpb9介导的TCR中的作用将通过控制其在细胞中的表达水平来研究。SPT4的缺失已被证明可以减轻Rad26对TCR的要求。Spt4在抑制Rpb9介导的TCR中的可能作用将通过在具有不同TCR亚通路的细胞中删除该基因来研究。多种成分可能参与两个TCR亚通路的调控。基因元件在调节中的作用将通过系统地创建这些元件的缺失和突变来检查。此外，转录水平和转录激活子Gal4如何参与调控也将被研究。

DESCRIPTION (provided by applicant): Organisms employ multiple conserved pathways to repair different types of DNA lesions. One important pathway is transcription coupled repair (TCR), which is responsible for rapid repair in the transcribed strand of an active gene. Defective TCR gives rise to the severe human disorder Cockayne's syndrome, and possibly other as yet unidentified diseases. Rpb9, a nonessential subunit of RNA polymerase II (Pol II), was recently found to mediate a TCR subpathway, which is superimposed on the Rad26 mediated TCR subpathway in Pol II transcribed genes. Rpb4, another nonessential subunit of Pol II, may play a dual role in regulating the two TCR subpathways, suppressing the Rpb9 mediated TCR and facilitating the Rad26 mediated TCR. The domains and critical residues of Rpb9 required for mediating TCR will be mapped by systematically deleting residues from the N- and C-termini, and by replacing certain critical residues. Other proteins involved in Rpb9 mediated TCR will be pulled down by epitope tagged Rpb9 or Rad14. The proteins pulled down will be identified by mass spectrometry analysis, and characterized genetically and biochemically. Yeast two hybrid system may also be utilized to screen potential proteins involved in Rpb9 mediated TCR. Covalent modifications of the largest subunit of Pol II in response to DNA damage may serve as TCR signals. Defl has been shown to be involved in ubiquitination and degradation of Pol II under certain TCR circumstances. The roles of Rpb9 and Defl in the covalent modifications of the largest subunit of Pol II will be examined by using Western blot analysis. The domains of Rpb4 required for suppressing Rpb9 mediated TCR will be mapped by systematically deleting residues from different regions of the protein. Rpb7 is an essential subunit of Pol II and forms complex with Rpb4. The role of Rpb7 in suppressing Rpb9 mediated TCR will be examined by controlling its expression levels in the cell. Deletion of SPT4 has been shown to alleviate the requirement of Rad26 for TCR. The possible role of Spt4 in suppressing Rpb9 mediated TCR will be studied by deleting the gene in cells with different TCR subpathways operative. Multiple components may be involved in the regulation of the two TCR subpathways. The roles of gene elements in the regulation will be examined by systematically creating deletions and mutations of these elements. Furthermore, how transcription levels and the transcription activator Gal4 may be involved in the regulation will also be investigated.