Pathways of mutagenesis at (GAA)n trinucleotide repeats in dividing and quiescent Saccharomyces cerevisiae
Neil, Alexander.
2018
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The goal of this
thesis is to examine how long repetitive DNA sequences comprised of (GAA)n trinucleotide
repeats lead to genetic mutations. Understanding the mechanisms of mutagenesis at these
sequences will aid in our understanding of the childhood-onset, neurodegenerative
disease Friedreich's Ataxia (FRDA), which results from the inheritance of abnormally
long (GAA)n repeats. In order to ... read moremeasure the frequency, rate and spectrum of mutations
at sequences within and around (GAA)n repeats, we have utilized genetically engineered,
selectable reporter systems that we integrated into the genome of the model organism
Saccharomyces cerevisiae. These constructs allow us to select for cells that acquire
mutations due to the presence of a long (GAA)n repeat by plating populations on media
that contain antibiotics. The thesis contains three major projects. First, we show that
mutations to the DNA replication protein Rad27 that inhibit its ability to appropriately
guide single-stranded DNA into its catalytic core results in a dramatic elevation in the
rate at which (GAA)n repeats expand. These expansions are primarily large-scale in
nature, suggesting that their mechanism precedes via a form of post-replicative DNA
repair known as template switching. Second, we demonstrate that deleting the yeast genes
RNH1 and RNH201, which encode for RNase H1 and the catalytic subunit of RNase H2
respectively, also leads to an increase in (GAA)n expansion rates. RNase H enzymes
specifically target and degrade the RNA component of RNA:DNA hybrids, which arise during
transcription and replication. Because this increase in repeat expansion rate in RNase H
deficient strains was only observed when repeats were transcribed, was independent of
the orientation of the repetitive sequence with respect to transcription and
replication, and was independent of the ability of (GAA)n repeats to serve as promoter
sequences, we concluded that expansion was instigated by the formation of a complex
non-B-DNA structure in which triplex H-DNA is stabilized by the formation of an RNA:DNA
hybrid (H-loop). Third, we examine the spectrum of deleterious mutagenesis at (GAA)n
repeats in quiescent cells. We show that the primary mutation type at (GAA)n repeats in
non-dividing cells is large-scale deletion events that are the result of inaccurate
non-homologous end joining following a mismatch repair-induced double strand break at or
near the repetitive sequence. These breaks can also lead to ectopic recombination events
that depend on the RAD51 and RAD52 genes. In msh3Δ strains, which do not form
breaks at the (GAA)n repeat, the predominant mutation type during quiescence is
excision-repair mediated iterative repeat expansions. Altogether, these works outline a
framework for mutagenesis at (GAA)n repeats whereby mutagenic outcomes are governed by
the cell-cycle, genotype and abnormal-structure formation at the repetitive
sequence.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Genetics.
Advisor: Sergei Mirkin.
Committee: Catherine Freudenreich, Mitch McVey, and Claire Moore.
Keywords: Genetics, and Molecular biology.read less - ID:
- fb494n48k
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