Role of One Double-Strand DNA Break in Yeast Meiosis

点击翻译按钮获取中文摘要

Meiotic recombination differs from mitotic recombination in many important respects. Meiotic recombination is initiated by double-strand breaks (DSB) made by the meiosis-specific Spo11 topoisomerase and requires at least nine other proteins to create the chromosomal environment in which these breaks occur. It is not known if these many proteins also dictate the outcome of Spo11-mediated gene conversions, which show a very high level of crossing-over relative to mitotic events. Meiotic crossing-over is also strongly influenced by at least two classes of proteins, the meiosis-specific Zip proteins that are components of the synaptonemal complex and the Mlhl-Msh4-Msh5 proteins. In contrast, mitotic gene conversions can be initiated by the site-specific HO endonuclease, which does not require the action of other proteins to make DSBs. In this project the HO endonuclease is expressed under the control of a meiosis specific promoter, so that it becomes possible to compare recombination events initiated by the same DSB in both meiosis and mitosis. A major goal of this work is to use this system to ask if the 9 other proteins needed to make meiotic DSBs are also needed to ensure a high proportion of crossing-over. Additionally, the system can be used to determine if the endonuclease can induce a high level of crossing-over in a "cold" region of chromosome III that represses normal Spo11-induced events. The second major goal of this work is to examine the control of meiotic crossing-over. An aspect of this will be to assess by cytological means if a single, HO-induced crossover is sufficient to promote equational chromosome segregation at the first meiotic division. This approach addresses the question whether such a reductional division will occur in the absence of SC, which appears to depend on the presence of many Spo11-mediated DSBs. The third objective is to use this unique DSB to investigate the molecular mechanism of recombination, with a goal of distinguishing if meiotic recombination, like HO-induced mitotic recombination, proceeds by a synthesis-dependent strand annealing mechanism. Finally, the system is used to investigate the important question of how one crossing-over event interferes with a crossover in an adjacent interval. It has recently been established that some chromosomal intervals do not actually exhibit any significant interference in Saccharomyces. Once appropriate intervals have been identified, one or two HO cleavage sites will be inserted to ask if HO-mediated events interfere with adjacent Spo11-induced crossovers and if two HO-induced crossovers also show interference.