The Advantage of Instability in Maintaining Function Within the Repetitive CTD of RNA Polymerase II
Abstract: The carboxyl-terminal domain (CTD) of RNA polymerase II is composed of a tandemly repeating 7 amino acid sequence that is conserved amongst eukaryotes. The DNA that encodes repetitive amino acid sequences is thought to be unstable and prone to both expansion and contraction of the DNA sequence, resulting in protein products of different lengths. Previous studies have shown mutations that ... read moreconfer a shorter CTD protein product impair growth, yet spontaneous suppressor mutations can arise in which the CTD coding region rearranges by expansion or contraction to restore growth. Our goal was to study this plasticity of the CTD by using a novel genetic reporter to measure the rate at which the CTD contracted to repair a series of premature stop codons. Specifically, we inserted stop codons in repeats 8-11, which produced a shortened protein product and resulted in diminished growth. The CTD was indeed able to contract and delete the stop codon containing repeats, and occasionally delete extra repeats in the contraction process. To explore the mechanisms involved in these contraction events, we measured mutation frequency in S. cerevisiae in which factors important for DNA repair had been deleted. Our results show that, unlike CTD expansion, which requires homologous recombination, contraction percentage was greatly increased in rad52Δ cells. We also show that the contraction mechanism does not involve Pol32p or Ku70p and is likely occurring through a Rad5p-mediated pathway. We have used this data to build a CTD contraction model wherein we propose that the DNA encoding the repetitive CTD region is unstable, allowing repeats with mutations to be deleted without compromising viability. Contractions are likely occurring through fork stalling and misalignments, and regions of microhomology around deletions indicate potential involvement of microhomology mediated end joining. These findings will be helpful in explaining the variation in CTD repeat number amongst individuals within a species, as well as a useful general model for the instability in genomic regions encoding repetitive amino acid sequences.
Thesis (M.S.)--Tufts University, 2016.
Submitted to the Dept. of Biology.
Advisor: Stephen Fuchs.
Committee: Mitch McVey, and Juliet Fuhrman.
Keywords: Biology, Molecular biology, and Genetics.read less