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Principal Investigator: Kathiresan Selvam
Organization: WASHINGTON STATE UNIVERSITY
Fiscal Year: 2024
Award: $244,750
Funding agency: National Institute of Environmental Health Sciences
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.
Terms: <Actinic Rays><Aging><Antibodies><BTF2 transcription factor><Baker's Yeast><Binding><Biochemistry><Biological Chemistry><Brewer's Yeast><Budding Yeast><C-terminal><Casein Kinase TS><Casein Kinase-2><Cell Body><Cells><Cockayne Syndrome><Complex><Coupled><Cyclobutane-Pyrimidine Dimers><DNA Damage><DNA Damage Repair><DNA Injury><DNA Repair><DNA lesion><DNA mapping><DNA-Dependent RNA Polymerase II><Data><Defect><Development><Drosophila><Drosophila genus><Elongation Factor><Endomycetales><Exhibits><Gene Down-Regulation><Gene Expression><Gene Transcription><GeneHomolog><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genome Stability><Genomic Stability><Health><Hereditary><Homolog><Homologous Gene><Homologue><Human><In Vitro><Inherited><Lesion><Maintenance><Mammalian Cell><Methods><Modern Man><Molecular><Molecular Interaction><Mutate><Mutation><Nerve Degeneration><Neuron Degeneration><Ortholog><Orthologous Gene><PH Domain><Pathway interactions><Patients><Phosphorylation><Phosphorylation Site><Play><Pleckstrin-Homology Domain><Polymerase><Progeria-Like Syndrome><Protein Kinase CK2><Protein Kinase CKII><Protein Phosphorylation><Proteins><Pyrimidine Dimers><RNA Expression><RNA Polymerase B><RNA Polymerase II><Role><S cerevisiae><S. cerevisiae><SEQ-AN><Saccharomyces cerevisiae><Saccharomycetales><Sequence Analyses><Sequence Analysis><Symptoms><Syndrome><TFIIH><Testing><Transcription><Transcription Elongation><Transcription Repression><Transcription-Coupled Nucleotide Excision Repair><Transcription-Coupled Repair><Transcriptional Elongation Factors><Transcriptional Repression><UV damage><UV induced><UV induced damage><UV light><UV radiation><UV radiation-induced><UV rays><Ubiquitilation><Ubiquitination><Ubiquitinoylation><Ultraviolet Rays><Unscheduled DNA Synthesis><Yeasts><basic transcription factor 2><casein kinase II><developmental><fruit fly><gene repair><gene repression><genome mutation><genome scale><genome wide analysis><genome wide studies><genome-wide><genome-wide analysis><genome-wide identification><genomewide><insight><neural degeneration><neurodegeneration><neurodegenerative><neurological degeneration><neuronal degeneration><pathway><prevent><preventing><recruit><repair><repaired><response><scaffold><scaffolding><social role><transcription factor IIH><transcription factor TFIIH><ubiquination><ubiquitin conjugation><ultra violet><ultra violet damage><ultra violet light><ultra violet radiation><ultra violet rays><ultraviolet><ultraviolet damage><ultraviolet induced><ultraviolet light><ultraviolet light induced><ultraviolet radiation><ultraviolet radiation-induced><yeast genetics><yeast genome>