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Melinda S. Borrie
B.S., Virginia Polytechnic Institute & State University - 2011
Thesis Advisor: Marc R. Gartenberg, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology
Rutgers School of Public Health
Thursday, April 13, 2017
Maintaining genome integrity during the process of cell division is aided by a ring-shaped protein complex known as cohesin. The complex binds chromatin topologically to mediate important nuclear functions including sister chromatid cohesion after DNA replication, double stranded DNA break repair through homologous recombination and the regulation of gene expression. In yeast, cohesin often associates with the bodies of inactive genes but transcriptional activation mobilizes the complex. This leads to accumulation of cohesin at the intergenic regions between convergently oriented genes. It is not clear whether transcription mobilizes cohesin by sliding complexes along DNA or if transcription displaces complexes from DNA, which then rebind at secondary locations.
Here, URA3 was used as a model system to study how cohesin arrives at a eukaryotic gene and what happens to the complex following transcription. To study cohesion, site-specific recombination was used to generate a pair of DNA circles from sister chromatids. Inclusion of URA3 in the circles caused cohesion but only when the gene was not induced. Cohesion of URA3 required a promoter-proximal poly(dA-dT) tract, a sequence motif commonly found at binding sites of the Scc2/4 cohesin loading complex. Chromatin Immunoprecipitation experiments revealed that not all cohesin that associates with DNA functions in cohesion. The data suggest that only functional cohesin complexes load onto URA3 via this poly(dA-dT) tract.
The DNA circle assay was also used to study how cohesive complexes are mobilized by transcription. Transcription of the locus was generally incompatible with its cohesion, causing increased turnover of the complex. Induction of transcription before circularization (when the template was linear) abolished cohesion. In contrast, cohesion persisted when transcription was induced after circularization (when the template was topologically closed). Insertion of a convergent gene downstream of URA3 also prevented transcription-driven loss of cohesion. Collectively, these data are consistent with transcription mobilizing cohesin by sliding the complexes along DNA.