Kong, Stephanie Ellen; (2001) RNA Polymerase II during Transcript Elongation: deaiing with DNA damage and staying phosphorylated. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis covers two topics related to transcript elongation in Saccharomyces cerevisiae - the regulation of the phosphatase Fcp1 and transcription-coupled DNA damage repair. Formation of RNA polymerase II (RNAPII) complexes throughout the transcription cycle is mediated in part by the phosphorylation state of the C-terminal domain (CTD) of the largest RNAPII subunit. Although a multitude of kinases can phosphorylate the CTD, currently only one CTD-specific phosphatase, Fcp1, has been identified. This work studies the possibility that Fcp1 might be associated with the elongating form of RNAPII. The phosphatase co-fractionates with RNAPII in association with the elongation factor Elongator. Furthermore, genetic studies show that a double mutant that carries a deletion of an Elongator gene as well as a temperature sensitive fcp1 mutation has a synthetic lethal phenotype at the permissive temperature. In vitro assays using crude extracts demonstrate that the CTD of RNAPII becomes dephosphorylated in a Fcp1-dependent manner. In contrast, in a reconstituted DNA-RNA-RNAPII system, the addition of the purified phosphatase does not stimulate such dephosphoryation. These results indicate a close relationship between Fcp1 phosphatase and the elongating form of RNAPII. Transcription-coupled DNA damage repair is a term applied to the preferential repair of DNA damage on the coding strand within active genes. The second half of this thesis describes the characterisation of nucleotide excision repair (NER) of the intrastrand 1,3-(pGpTpG)-cisplatin lesion in Saccharomyces cerevisiae as well as an attempt to reconstitute a transcription-coupled NER reaction (TC-NER) in vitro. Using modified yeast extracts, the excision products of the above lesion by NER were found to be between 23 and 26 nucleotides long, via incisions around the 15th phosphodiester bond 3' and 7th the phosphodiester bond 5' of the damage. The attempt to reconstitute TC-NER in vitro was hindered by difficulties with the transcription substrate and the functional instability of purified NER proteins.

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