Author ORCID Identifier
Date of Graduation
Microbiology and Molecular Genetics
Doctor of Philosophy (PhD)
Nayun Kim, PhD
Theresa M. Koehler, PhD
Grzegorz Ira, PhD
Lei Li, PhD
Jiqiang Ling, PhD
Recombination and mutagenesis are elevated by high levels of transcription. The correlation between transcription and genome instability is largely explained by the topological and structural changes in DNA and the associated physical obstacles generated by the transcription machinery. However, such explanation does not directly account for the unique types of mutations originating from the non-canonical residues such as uracil, which are also elevated at highly transcribed regions. Apurinic/Apyrimic or Abasic (AP) sites derived from uracil excision, accumulate at a higher rate in actively transcribed regions of the genome in S. cerevisiae and are primarily repaired by base excision repair (BER) pathway. I have demonstrated that transcription-coupled nucleotide excision repair (NER) pathway can functionally replace BER to repair those AP sites located on the transcribed strand much like the strand specific repair of UV-induced pyrimidine dimers.
This thesis reveals that the DNA composition can be modified to include higher uracil-content through the non-replicative, repair-associated DNA synthesis. I show here a positive correlation between the level of transcription and the density of uracil residues in the yeast genome indirectly through the mutations generated by the glycosylase that excise undamaged cytosine as well as uracil. The higher uracil-density at actively transcribed regions is confirmed by the long-amplicon qPCR analysis. I also show that the uracil- associated mutations at highly transcribed regions are elevated by the induced DNA damage and reduced by the overexpression of a dUTP-catalyzing enzyme, Dut1, in G1- or G2-phases of the cell cycle.
Additional roles of transcription elongation factor Dst1 and RNAPII degradation factor Def1 in AP induced transcription arrest is also revealed. I report that Def1 directs NER to AP lesions on the transcribed strand of an actively transcribed gene but that its function is dependent on metabolic state of the yeast cells. I additionally show that Dst1, a homolog of mammalian transcription elongation factor TFIIS, interferes with NER-dependent repair of AP lesions while suppressing homologous recombination pathway.
In summary, this thesis elucidates a novel mechanism of introducing uracil into DNA during damage-induced repair synthesis and provides further insights onto how AP sites on the transcribed DNA strand are repaired.
Uracil, Transcription, BER, TC-NER, Yeast, Mutations, Repair Synthesis, DNA damage