| DNA topoisomerase II (Topo II) is a conserved enzyme that allows double-stranded DNA molecules to pass through one another. This reaction is required for many aspects of DNA metabolism, particularly chromosome segregation in mitosis. This dissertation presents evidence that Top2, the Topo II enzyme in the budding yeast Saccharomyces cerevisae, acts at regions called centromeres to promote chromosome segregation, and that a post-translational modification known as SUMO modification controls these aspects of Top2 function.;Centromeres are assembly sites for kinetochores, which mediate attachment to the mitotic spindle. As kinetochores on chromosome pairs attach to the spindle they are pulled towards opposing spindle poles, generating mechanical tension and stretching of centromeric DNA. In catalytically inactive top2-4 and SUMO modification-resistant top2-SNM mutants both the distance and frequency of centromere stretching is increased. Using a novel assay, we found that increased stretching in top2 mutants corresponds with overwinding of centromeric DNA. These observations, and other data, suggest centromere stretching induces topological strain. This is relaxed by Top2 to promote a compact centromere organization that resists the pulling force of the spindle.;A second set of observations reveals a novel role for Top2 in monitoring kinetochores. During attachment, an error correction mechanism releases kinetochores that are not placed under tension; detachment is mediated through a centromere-localized protein kinase called Ipl1. One model for error correction is that centromere stretching allows correctly tensed kinetochores to be pulled away from Ipl1, allowing them to become resistant to detachment. In top2-4 and top2-SNM mutants, however, tension-deficient kinetochores are not disconnected, suggesting altered centromere compliance may enable weakened kinetochores to stretch centromeric chromatin and escape Ipl1.;A final component examines how SUMO modification promotes Top2 function. As evaluated by florescence recovery after photobleaching, SUMO modification plays an important role in allowing Top2 to exchange rapidly throughout the nucleus. To explain this, I propose that in certain chromatin environments, such as centromeres under tensile strain, a fraction of Top2 becomes stalled in catalysis. SUMO modification may target this population to promote catalytic turnover. Defects in this response may reduce Top2 catalytic efficiency at centromeres, explaining defects in top2-SNM mutants. |
