The long term objectives are to identify the mechanisms eukaryotic cells employ to overcome blocks to RNA polymerase II (Pol II) transcription conferred by DNA lesions in the template strand.
The specific aims of this proposal are to determine the roles of the yeast Rad26, Rad28, and Rad2 proteins, and of TFIIH, particularly its Rad3 and Rad25 DNA helicase subunits, in transcription elongation on undamaged and damaged DNAs, and in transcription-coupled DNA repair.
In Specific Aim 1, the involvement of the RAD26, RAD28, RAD2, RAD3, and RAD25 genes in transcription elongation in undamaged cells will be examined by determining the effects of mutations in these genes on Pol II transcription in yeast cells grown in the presence of 6-azauracil (6AU), and by determining the in vivo occupancy of promoters and open reading frames (ORFs) of various genes by the Rad26, Rad28, Rad2, Rad3, and Rad25 proteins by chromatin immunoprecipitation (CHIP) assays in yeast cells treated with 6AU.
In Specific Aim 2, biochemical studies will be done to examine the effects of purified Rad26, Rad28, Rad2, and TFIIH on Pol II transcription elongation through intrinsic arrest sites on undamaged DNA templates.
In Specific Aim 3, the interactions of the Rad2, Rad26, and Rad28 proteins with Pol II will be examined.
In Specific Aim 4, the involvement of Rad26, Rad28, Rad2, and TFIIH in promoting Pol II transcription through damaged bases and through abasic sites will be studied by genetic and biochemical means. To provide evidence for the in vivo roles of these genes in the transcriptional bypass of DNA lesions, Pol II transcription will be examined in the various rad mutant strains that are also deleted for the genes that function in the removal of damaged bases or abasic sites, and treated with the alkylating agent MMS. The presence of the Rad26, Rad28, Rad2, Rad3, and Rad25 proteins with elongating Pol II will be analyzed by ChIP analyses in cells treated with MMS. Biochemical studies will be done with purified Rad26, Rad28, Rad2, and TFIIH to examine their role in promoting Pol II transcription through a thymine glycol and an abasic site.
In Specific Aim 5, the hypothesis that after promoting the forward translocation of Pol II through certain DNA lesions, the Rad26, Rad28, Rad2 and TFIIH proteins remain bound at the lesion site and provide the nucleation site for the subsequent assembly of other repair factors, thereby enabling the preferential repair of DNA lesions from the transcribed strand, will be tested. Mutations in the human counterparts of yeast genes being studied here can cause Cockayne syndrome (CS); the proposed studies in yeast should yield a better understanding of the underlying basis of the severe growth and developmental defects in CS.
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