Chromosome dynamics of the early meiotic cell cycle in S. cerevisiae

In every cell cycle the genetic material must be duplicated and transmitted to the daughter cells. Meiosis is a developmental program that allows a diploid cell to produce haploid progeny. The reduction in chromosome number obtained during meiosis requires specialized mechanisms that are absent during the canonical mitotic cell cycle. Although previous studies found strong similarities between pre-mitotic and pre-meiotic DNA replication, pre-meiotic S phase is longer than pre-mitotic S phase, suggesting that meiosis-specific events regulate the rate of DNA replication. Additionally, after DNA replication, homologous recombination is initiated by the introduction of hundreds DNA double-strand breaks (DSBs) into the genome to produce physical DNA exchnges, or crossovers, between homologous chromosomes. To investigate the chromosome dynamics of the early meiotic cell cycle, I performed comprehensive analysis of pre-meiotic DNA replication and DSB formation in budding yeast. Genome-wide studies of pre-meiotic DNA replication confirmed that the same replication origins are selected and activated in pre-meiotic and pre-mitotic cells, although replication was delayed at a large number of origins. These results indicate that the regulation of DNA replication is similar in the meiotic and mitotic cell cycles, but that the replication-timing program differs. Elimination of meiosis-specific cohesion or homologous recombination had no effect on the number or identity of early pre-meiotic origins. Analysis of cells sporulated in the presence of the replication inhibitor HU revealed a Cln3-dependent inhibition of meiotic entry. To map the locations of meiotic DSBs, I developed a method to detect meiotic ssDNA. Examination of the sites of ssDNA enrichment indicated that DSBs occur mainly in the promoters of active genes, consistent with previous studies of individual DSB sites. Global analysis of the most common DSB sites revealed a non-random distribution of DSB "hotspots. " In particular, DSB hotspots are over-enriched close to chromosome ends, which could explain why small chromosomes have a higher DSB density than large chromosomes. This mechanism could help ensure that all homologous chromosomes receive at least one crossover and segregate properly in meiosis. These studies also indicated that suppression of recombination at telomeres, centromeres and around the rDNA occurs by 3 distinct mechanisms. Thesis advisor: Stephen P. Bell, Professor of Biology To Sister, Anjakat and Menalie, for their inspiration and support Acknowledgements This work could not have been completed without the help and support of a large number of people. Thank you to the past and present members of the Bell Lab family for creating a stimulating environment for scientific discovery and extracurricular hobbies. I am also indebted to past and present members of the Amon lab, particularly those in the meiosis group, for providing me with reagents, protocols, ideas and genuine friendship. Thank you to Andreas, as our collaboration has been great fun and the kind of success that the faculty told us would magically happen if we spent enough time together in the Pit (and Starbucks). My classmates in Biograd 2000 have become friends and colleagues in pursuits inside the lab, as well as dim sum, supper club, girls' night, river walks etc. My friends and family outside of MIT have constantly reminded me that I am part of a larger world that requires an excess of love, patience and creative vision to go 'round. I cannot thank my roommates, sister, Phil and Anna, enough for being there for me through this process as only the best of friends and family can. I am extremely grateful to past and present members of my thesis committee, Anja-Katrin Bielinsky, Frank Solomon, Terry Orr-Weaver and Rick Young, who have always imparted scientific and life advice when it was needed. I would like to specially acknowledge Angelika Amon, who has supported me ceaselessly as a mentor, second advisor, role model and friend. Thank you especially to Steve, who, from start to finish, entrusted me with the means and freedom to follow my whims, wherever they took us. I have enjoyed his sound advice, stimulating scientific discussions, ability to be understanding, and generous nature, which make the Bell Lab a great place to work.