A central problem in biology is how genetic material is organized and inherited during cell divisions without loss of information. The conserved protein complex cohesin functions to ensure chromosomes are partitioned equally when cells divide. Cohesin mediates sister chromatid cohesion, chromosome condensation, efficient repair of DNA damage and regulation of transcription. The activity by which cohesin performs these functions is through DNA tethering. Tethering can occur between two sister chromatids or within a single chromatid to bring together two separate positions along its length. Within this dissertation, I will describe novel insights into the mechanism by which cohesin performs these critical activities on chromosomes. I chose to study this problem in the budding yeast Saccharomyces cerevisiae because of the unparalleled genetic tools available in this model eukaryote.

Cohesin consists of four core subunits referred to in yeast as Mcd1p, Scc3p, Smc1p, and Smc3p, whereas spatiotemporal control of cohesin activity depends on auxiliary factors. To better understand cohesin architecture and regulation, I performed a genetic screen to identify novel mutants of the Smc3p subunit. Characterization of these mutants has revealed functions for previously undescribed regions of Smc3p and new activities for previously described domains that further our understanding of this complex molecular machine. First, I describe a series of mutations at the interface between Smc3p and Mcd1p termed the DNA “exit gate”. This investigation revealed an unexpected role for this interface in loading cohesin on chromosomes. Second, I reveal a novel role for the Smc3p hinge domain in cohesion maintenance that is independent of its ability to stably bind chromosomes. This discovery lends additional weight against a simple “embrace” model for DNA tethering by cohesin still supported by many in this field. Moreover, this observation suggests that communication between the Smc3p hinge, Mcd1p, and regulator Pds5p is required for cohesion maintenance. Finally, I show that a specific region in the middle of the cohesin ring is critical for a step in the process of cohesin loading onto chromosomes. This result is surprising since it suggests that a contortion of the cohesin ring is critical for it to productively bind chromosomes. In sum, this dissertation advances our understanding of the mechanism by which cohesin functions and supports a variation of the “handcuff” model in which an activity after DNA binding is required for cohesin to achieve tethering.

During cellular division, chromosomes undergo a series of cytological events that allow for the maintenance and inheritance of genetic information from one generation to the next. While the necessity of these morphological changes has been appreciated for over a hundred years, we still have but a rudimentary understanding of the basic principles underlying how chromosomes are formed.

Over the past two decades, we have gained an appreciation for the integral role of two related, multi-subunit Structural Maintenance of Chromosomes (SMC) protein complexes, cohesin and condensin, play in chromosome condensation. The necessity of both protein complexes is particularly evident in the budding yeast, Saccharomyces cerevisiae, where loss of either cohesin or condensin leads to cytologically indistinguishable loss of chromosome condensation.

In this dissertation, I address some of the gaps in our knowledge of the mechanisms governing mitotic chromosome condensation. This body of work was sparked by the fundamental question of how condensin and cohesin orchestrate their functions to ensure that chromosome condensation occurs in a tightly regulated spatio-temporal manner. To that end, we used a combination of yeast genetics, cytology, sequencing and chromosome conformation capture methodologies to dissect the relative contributions of cohesin and condensin to mitotic chromosome condensation.

First, we describe the role of a kinase, Polo (Cdc5), as part of an interactome of early condensation factors along with cohesin and condensin. In particular, we describe how cohesin modulation of this kinase may impact condensin function. Next, we utilize a chromosome conformation capture technique, Micro-C, to elucidate features of yeast mitotic structures at nucleosome resolution. There, we also begin to dissect the contributions of mitotic factors in these structures. Lastly, we investigate the molecular defects of a unique cohesin mutant that allow us to dissect cohesin’s role in condensation.

Altogether, these findings reshape our understanding how higher order chromosome structures are formed and inspire us to reevaluate some of our fundamental assumptions about how SMCs coordinate their function. Additionally, this work highlights how yeast can serve as a genetically-tractable model for studying the formation of chromosome loops.

R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in budding yeast, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. Our results indicate that persistent R-loops in the rDNA induce damage that is slowly repaired by break-induced replication (BIR). Furthermore, R-loop induced BIR at the rDNA leads to lethal repair intermediates when RNA polymerase I elongation is compromised. We present a model to explain how removal of R-loops by RNase H is critical in ensuring the efficient repair of R-loop induced DNA damage by pathways other than BIR.