Transcription Coupled DNA Repair in S. cerevisiae

酿酒酵母中的转录耦合 DNA 修复

• 批准号: 6820052
• 负责人: Shisheng Li
• 金额: $ 1.17万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2004-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820052
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; Saccharomyces cerevisiae; fungal genetics; fungal proteins; genetic regulation; genetic regulatory element; genetic transcription; intermolecular interaction; mass spectrometry; molecular assembly /self assembly; phosphorylation; posttranslational modifications; protein structure function; ubiquitin

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.

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。一个重要的途径是转录偶联修复（TCR），其负责活性基因的转录链中的快速修复。有缺陷的TCR引起严重的人类疾病Cockayne综合征，以及可能的其他尚未确定的疾病。Rpb 9是RNA聚合酶II（Pol II）的一个非必需亚基，最近被发现介导TCR亚通路，其叠加在Pol II转录基因中的Rad 26介导的TCR亚通路上。Pol II的另一个非必需亚基Rpb 4可能在调节两种TCR亚通路中发挥双重作用，抑制Rpb 9介导的TCR和促进Rad 26介导的TCR。介导TCR所需的Rpb 9的结构域和关键残基将通过从N-和C-末端系统地缺失残基以及通过替换某些关键残基来作图。参与Rpb 9介导的TCR的其他蛋白质将被表位标记的Rpb 9或Rad 14拉下。被拉下来的蛋白质将通过质谱分析进行鉴定，并在遗传和生物化学上进行表征。酵母双杂交系统也可用于筛选参与Rpb 9介导的TCR的潜在蛋白。Pol II最大亚基对DNA损伤的反应中的共价修饰可以作为TCR信号。已显示Defl在某些TCR情况下参与Pol II的泛素化和降解。Rpb 9和Defl在Pol II的最大亚基的共价修饰中的作用将通过使用Western印迹分析来检查。抑制Rpb 9介导的TCR所需的Rpb 4的结构域将通过系统地删除来自蛋白质不同区域的残基来定位。Rpb 7是Pol II的一个必需亚基，与Rpb 4形成复合物。Rpb 7在抑制Rpb 9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查。已显示缺失SPT 4可减轻TCR对Rad 26的需求。Spt 4在抑制Rpb 9介导的TCR中的可能作用将通过在具有不同TCR子途径的细胞中缺失该基因来研究。多种组分可能参与两种TCR子途径的调节。基因元件在调控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因子Gal 4如何参与调控也将被研究。