| Summary: | Nucleotide excision repair (NER) is a multi-step cellular process that removes bulky and/or helix-distorting DNA lesions, such as UV induced cyclobutane pyrimidine dimers (CPDs) and bulky chemical adducts. Transcription coupled repair (TCR) is a subpathway of NER dedicated to rapid removal of lesions in the transcribed strand of actively transcribed genes. The TCR mechanism in bacteria has been relatively well elucidated. However, TCR in eukaryotic cells appears to be extremely complicated. The exact nature of the TCR signal and the mechanism of the transcription-repair coupling have been long-standing enigmas. This dissertation focused on how the TCR repressors and facilitators interplay with RNA polymerase II (RNAP II) to carry out TCR in yeast Saccharomyces cerevisiae.
By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, we mapped interactions between Spt5 and RNAP II in S. cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR. We also demonstrated that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced CPDs, increases survival of UV irradiated yeast cells but attenuates TCR. In contrast, G730D mutation of Rpb1, which abolishes transcription bypass of CPDs, enhances TCR. Our findings suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. Sen1 is an RNA/DNA helicase that has been shown to mediate termination of noncoding RNAs and some mRNAs. Like deletion of Rad26 or Rpb9, the Sen1 N-terminal deletion (1-975 residues) increases the UV sensitivity of the GGR-deficient cells. Moreover, the Sen1 N-terminal deletion decreases TCR in rad7Δ and rad7Δ rad26Δ cells but not that in rad7Δ rpb9Δ cells. Our findings suggest that the N-terminal domain of Sen1 contributes to Rad26-independent TCR.
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