Role of One Double-Strand DNA Break in Yeast Meiosis

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Haber 9723086 Meiotic recombination is fundamental in the generation of diversity in people and in most other eucaryotes. Crossing-over between homologous chromosomes also plays a key role in proper chromosome segregation. The yeast Saccharomyces cerevisiae has provided a powerful model system to analyze these events by a combination of genetic, molecular biological and cytological techniques. Much progress has been made in understanding how meiotic recombination occurs and how recombination is related to the formation and function of the synaptonemal complex (SC). One central unanswered question is: how does meiotic recombination differ from mitotic recombination and to what extent this difference can be assigned to: a) the way double-strand breaks are created, b) the influence of the synaptonemal complex on gene conversion and crossing-over, or c) the expression of meiotic-specific recombination genes? By the meiotic expression of the site-specific HO endonuclease, under the control of the meiotic-specific SPOI3 promoter, it is now possible to induce a single HO double-strand break (DSB) in meiotic cells and to compare these events with those initiated by HO in mitotic cells. This provides, for the first time, a way to compare directly the recombination events initiated by the same DNA cleavage in these two cell types. Moreover, this system provides a way to examine recombination in spo11 or rad50 or other mutant meiotic cells that are incapable of creating normal meiotic DSBs and thus are incapable of initiating normal meiotic recombination. Consequently the effects of these mutations on later steps in meiotic recombination can be assessed, for the first time, by inducing a DSB with the HO endonuclease. Southern blot analysis revealed that SPO13::HO-induced events appear at the same time as normal events. A most striking result was obtained from a cytological analysis of spol3 red50 cells that cannot induce normal DSBs. Surprisingly, the creation of an HO-induced DSB at one site on one chr omosome triggered the formation of axes containing the synaptonemal complex protein, Zip1p, along many chromosomes. This suggests that the creation of DSBs along a chromosome is not necessary for the formation of the synaptonemal complex along that chromosome, and that formation of the SC is triggered in response to the detection of a single DSB. A major focus of the work is to understand in detail how the formation of the SC is triggered. Experiments to determine if SC formation initiated by an HO DSB depends on subsequent recombination events carried out by gene products such as RadS1, Rad52 and Dmc1are being carried out. Second, the role of mutations known to cause mitotic cell cycle delay in response to DNA damage (RAD9, RAD17, RAD53, TEL1) will be assessed for their role in triggering SC formation after an HO DSB in meiosis. Finally, HO induced DSBs will be used to determine if a single DSB is sufficient to cause homologous chromosome synapsis, or whether HO-induced SC formation occurs between nonhomologous chromosomes. Finally, an experimental system is being carried out to determine if one HO-induced DSB per chromosome is effective in directing proper chromosome segregation on chromosomes that lack any other crossover events.