Transcription Coupled DNA Repair in S. cerevisiae

描述(申请人提供)：生物体使用多种保守途径来修复不同类型的DNA损伤。一个重要的途径是转录偶联修复(TCR)，它负责对活性基因转录后的链进行快速修复。TCR缺陷会导致严重的人类疾病Cockayne综合征，可能还会导致其他尚未确定的疾病。Rpb9是RNA聚合酶II(Pol II)的一个非必需亚基，最近被发现在Pol II转录的基因中介导TCR亚途径，该亚途径叠加在Rad26介导的TCR亚途径上。Rpb4是Pol II的另一个非必需亚基，可能具有调节两条TCR亚通路、抑制Rpb9介导的TCR和促进Rad26介导的TCR的双重作用。通过系统地删除N-末端和C-末端的残基，并替换某些关键残基，将定位介导TCR所需的Rpb9结构域和关键残基。Rpb9介导的TCR中的其他蛋白将被标记为Rpb9或Rad14的表位拖拉。被拉下的蛋白质将通过质谱分析进行鉴定，并从遗传和生化方面进行表征。酵母双杂交系统也可用于筛选Rpb9介导的TCR的潜在蛋白。Pol II最大亚基对DNA损伤的共价修饰可能是TCR信号。研究表明，在某些TCR环境下，Defl参与了Pol II的泛素化和降解。Rpb9和Def1在POL II最大亚基的共价修饰中的作用将通过蛋白质印迹分析来检测。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定位。Rpb7是POL II的一个重要亚基，与Rpb4形成复合体。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检验。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。这两条TCR亚通路的调控可能涉及多个成分。基因元件在调控中的作用将通过系统地产生这些元件的缺失和突变来研究。此外，转录水平和转录激活因子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.