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The Impact of Homologous Recombination on Silent Chromatin in Saccharomyces cerevisiae

  • Author(s): Sieverman, Kathryn Jeanne
  • Advisor(s): Rine, Jasper
  • et al.
Abstract

Specialized chromatin domains repress transcription of genes within them and present a barrier to many DNA-protein interactions. Silent chromatin in the budding yeast Saccharomyces cerevisiae, akin to heterochromatin of metazoans and plants, is imparted by the Silent Information Regulation (SIR) proteins, Sir1-Sir4. Silencing is well described at the HML and HMR loci in S. cerevisiae, which harbor un-expressed copies of the genes necessary for mating-type identity. SIR-silenced chromatin inhibits transcription of PolII- and PolIII-transcribed genes, yet somehow grants access to proteins necessary for DNA transactions such as replication and homologous recombination. Homologous recombination within silent chromatin is a key aspect of yeast biology, as the process of mating-type switching depends upon it. Mating-type switching initiates with a programmed double-strand break at the MAT locus, which then accesses either HML or HMR to template homologous recombination and repair the double-strand break. Whether homologous recombination impacts the stability of silent chromatin at HML or HMR was not known when I began this study.

To investigate the impact of homologous recombination on silent chromatin, I developed an assay to detect even transient changes in the dynamics of transcriptional silencing at HML after it served as a template for homologous recombination. Homologous recombination specifically targeted to HML often led to transient loss of transcriptional silencing at HML. Interestingly, many cells could template homology-directed repair at HML without an obligate loss of silencing, even in recombination events with extensive gene conversion tracts. Thus, recombination with HML led to two distinct outcomes, one in which silent chromatin was disrupted enough to permit transcription, and one in which silencing persisted despite the presence of the homologous recombination machinery.

In the cells that experienced silencing loss following recombination, transcription persisted for two to three hours after all double-strand breaks were repaired. mRNA levels from cells that experienced recombination-induced silencing loss did not approach the amount of mRNA seen in cells lacking transcriptional silencing. Thus, silencing loss at HML after homologous recombination was short-lived and limited.

I explored the possibility that chromatin-remodeling complexes known to play a role in homology-directed repair might affect the stability of silent chromatin after homologous recombination. Analysis in a background mutant for the SWI/SNF or INO80 chromatin-remodeling complexes revealed no clear role for nucleosome remodeling by these complexes in influencing the likelihood of silencing loss at HML associated with homologous recombination.

In the process of investigating post-recombination silencing loss, I found that the founder cell of a colony experienced a different likelihood of both silencing loss and gene conversion from its progeny. This manifested as colonies grown from single cells bearing cells that both maintained and lost silencing, as well as cells that both did and did not show evidence of homologous recombination. Thus, differences in both phenotype and genotype existed between a founder cell and its early progeny after double-strand break induction and repair.

The work in this dissertation has laid the foundational observations for further dissecting the processes that can disrupt what is otherwise a remarkably stable repressive chromatin structure.

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