Role of One Double-Strand DNA Break in Yeast Meiosis

减数分裂重组是人类和大多数其他真核生物多样性产生的基础。同源染色体间的交叉在染色体分离中也起着关键作用。酵母（Saccharomyces cerevisiae）通过遗传学、分子生物学和细胞学技术的结合，为分析这些事件提供了一个强大的模型系统。在了解减数分裂重组如何发生以及重组与突触复合体（SC）的形成和功能的关系方面取得了很大进展。一个悬而未决的核心问题是：减数分裂重组与有丝分裂重组有何不同，这种差异在多大程度上可以归结为：a)双链断裂产生的方式，b)突触复合体对基因转换和交叉的影响，或c)减数分裂特异性重组基因的表达？在减数分裂特异性SPOI3启动子的控制下，通过位点特异性HO内切酶的减数分裂表达，现在可以在减数分裂细胞中诱导单个HO双链断裂（DSB），并将这些事件与有丝分裂细胞中HO引发的事件进行比较。这首次提供了一种方法，可以直接比较这两种细胞类型中由相同DNA切割引发的重组事件。此外，该系统提供了一种方法来检查spo11或rad50或其他突变减数分裂细胞中的重组，这些细胞不能产生正常的减数分裂dsb，因此不能启动正常的减数分裂重组。因此，这些突变对减数分裂重组后期步骤的影响可以首次通过诱导带有HO内切酶的DSB来评估。Southern blot分析显示，SPO13:: ho诱导的事件与正常事件同时发生。从不能诱导正常dsb的spol3red50细胞的细胞学分析中获得了一个最惊人的结果。令人惊讶的是，在一个chr染色体上的一个位点上，ho诱导的DSB的产生触发了沿许多染色体形成包含突触复合蛋白Zip1p的轴。这表明沿染色体产生的DSB对于沿染色体形成突触复合体并不是必需的，SC的形成是在检测到单个DSB时触发的。这项工作的一个主要重点是详细了解SC的形成是如何触发的。正在进行实验，以确定由HO DSB启动的SC形成是否取决于随后由基因产物（如RadS1、Rad52和dmc1）进行的重组事件。其次，在DNA损伤的反应中，已知引起有丝分裂细胞周期延迟的突变（RAD9, RAD17, RAD53, TEL1）在减数分裂HO DSB后触发SC形成中的作用将被评估。最后，HO诱导的DSB将用于确定单个DSB是否足以引起同源染色体突触，或者HO诱导的SC形成是否发生在非同源染色体之间。最后，一个实验系统正在进行，以确定是否一个ho诱导的DSB每条染色体是有效地指导正确的染色体分离，染色体缺乏任何其他交叉事件。

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.