Saturday, September 7, 2024
9/7/2024
ABSTRACT Transcription coupled-nucleotide excision repair (TC-NER) plays a critical role in the maintenance of genome stability by removing DNA lesions that would otherwise block RNA polymerase II (Pol II) transcription. The importance of TC-NER to human health is highlighted in Cockayne syndrome (CS), in which an inherited genetic defect in a TC-NER factor (CSA or CSB) results in a severe neurodegeneration, rapid aging, and UV sensitivity syndrome. Recently, a new TC-NER factor known as ELOF1 in humans and Elf1 in yeast has been identified. ELOF1/Elf1 are required for efficient TC-NER in both yeast and human cells. In human cells, ELOF1 functions to recruit the key TC-NER factor UVSSA, which is required for TFIIH recruitment, by promoting RNA polymerase II ubiquitination. However, yeast and many other species lack UVSSA, so the function of Elf1 in promoting TC-NER in these species is unclear. Our preliminary data indicate that a unique C-terminal domain (CTD) present in Elf1 plays a critical role in TC-NER by recruiting TFIIH. Our preliminary data also suggest the hypothesis that the Pol II elongation factors Spt4/Spt5 (Spt4/5), which normally function to repress TC-NER, do so by inhibiting the Elf1 CTD. We hypothesize that eviction of Spt4/5 from the Pol II elongation complex (EC) by the CSB homolog Rad26 initiates repair by releasing the Elf1 CTD so it can recruit TFIIH. In Aim I, we will use a combination of yeast genetics, genome-wide mapping of DNA damage and repair, and in vitro biochemistry to test this hypothesis. Cyclobutane pyrimidine dimer-sequencing (CPD-seq) method will be used to investigate the interplay of the Elf1 CTD and Spt4/5 in genome-wide repair of UV damage in yeast and characterize key functional residues in the CTD. We will also use in vitro protein interaction studies to determine the key CTD residues in Elf1 that are required to bind and recruit TFIIH, and test whether this interaction is inhibited by Spt4/5. Finally, we will test the hypothesis that Drosophila homologs of Elf1 are responsible for TC-NER activity in this species, despite the absence of Drosophila homologs of CSA, CSB, or UVSSA. In Aim II, we will investigate the role of phosphorylation of the Elf1 CTD in regulating its activity in TFIIH recruitment and TC-NER. Previous studies and our preliminary data indicate that the Elf1 CTD is phosphorylated at multiple residues, in many cases by casein kinase II (CKII). In this aim, we will systematically mutate Elf1 phosphorylation sites and determine their impact on TC-NER. In parallel, we will screen for new UV-induced phosphorylation sites in the Elf1 CTD. We will also determine whether CKII phosphorylation of the Elf1 CTD in vitro regulates its binding to TFIIH and/or Spt4/5 and determine the potential role of CKII in TC-NER. These studies should elucidate a new molecular mechanism by which CSB/Rad26-dependent modulation of the Pol II EC regulates TFIIH recruitment and TC-NER.
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