Nucleotide excision repair (NER), a “cut and patch” mechanism, has two subpathways, namely transcription coupled repair (TCR) and global genome repair (GGR). TCR rapidly removes UV photoproduct (UV PP) in the transcribed strand (TS) of an actively transcribed gene, whereas GGR removes those DNA lesions throughout the genome. This dissertation mainly focused on identifying novel factors that function in NER and investigating the potential molecular mechanisms underlying initial damage recognition and downstream processes in NER. We identified Rpb7, a subunit of RNA polymerase II, represses TCR via its multiple regions. Specifically, the conserved region of Rpb7 that interacts with the KOW3 domain of Spt5 represses TCR, which may share the same common mechanism as Spt5. On the other hand, regions of Rpb7 that interact with Rpb4 and/or the core RNAPII repress TCR, which may be largely independently of Spt4/Spt5. Tfb1, a subunit of TFIIH, contains a Pleckstrin homology domain (Tfb1-PHD). The two PHD binding segments (Rad2-PB1 and Rad2-PB2) of Rad2, and one PHD-binding segment (PB) of Rad4 have been reported to interact with Tfb1-PHD in vitro. It remains unknown whether/how these interactions occur and contribute to TCR and/or GGR in the cell. We found that only Rad2 PB1 interacts with Tfb1 PHD in yeast cells and the interaction was enhanced during NER. The enhanced interaction is dependent on Rad4. In contrast, Rad2 PB2 inhibits the interaction between Rad2 PB1 and Tfb1 PHD when NER is inactive. By functioning with Tfb1 PHD, both Rad2 PB1 and PB2 contribute to NER. Rad4 PB constitutively interacts with Tfb1 PHD and this interaction plays more significant roles in NER. Our findings suggest that Rad4 and Tfb1 PHD function together to initiate damage recognition in NER. Histone chaperone Spt6 is important for chromatin remodeling, transcription, and replication. Here, we found that spt6-1004 mutant derepresses Rad26 independent TCR but impairs GGR, whereas the truncation of Spt6 tSH2 domain attenuates Rad26 independent TCR, particularly in highly transcribed gene YEF3. On the other hand, both mutants significantly decrease UV induced mutagenesis, suggesting Spt6 may also function in DNA damage tolerance.
Gong, Wenzhi, "Regulation of Nucleotide Excision Repair" (2023). LSU Doctoral Dissertations. 6207.
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