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.
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