Role of One Double-Strand DNA Break in Yeast Meiosis

一条双链 DNA 断裂在酵母减数分裂中的作用

• 批准号: 9724086
• 负责人: James Haber
• 金额: $ 28.5万
• 依托单位: Brandeis University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724086&HistoricalAwards=false
• 关键词: Role; Double; Strand; DNA; Break

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.

Haber 9723086减数分裂重组是人类和大多数其他真核生物产生多样性的基础。同源染色体之间的交换在染色体分离中也起着关键作用。酿酒酵母为结合遗传学、分子生物学和细胞学技术分析这些事件提供了一个强有力的模型系统。在了解减数分裂重组如何发生以及重组与联会复合体（SC）的形成和功能的关系方面取得了很大进展。一个尚未回答的中心问题是：减数分裂重组与有丝分裂重组有何不同，以及这种差异在多大程度上归因于：a）双链断裂的产生方式，B）联会复合体对基因转换和交换的影响，或c）减数分裂特异性重组基因的表达？ 通过在减数分裂特异性SPOI3启动子的控制下的位点特异性HO内切核酸酶的减数分裂表达，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由HO引发的事件进行比较。这首次提供了一种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的方法。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通过用HO内切核酸酶诱导DSB来首次评估。Southern印迹分析显示，SPO13：：HO诱导的事件与正常事件同时出现。一个最引人注目的结果是从spol 3 red50细胞的细胞学分析，不能诱导正常的DSB。令人惊讶的是，在一个染色体上的一个位点上产生HO诱导的DSB触发了包含联会复合体蛋白Zip1p的轴的形成，沿着许多染色体。这表明，沿着染色体沿着产生DSB对于沿着该染色体形成联会复合体沿着不是必需的，并且SC的形成是响应于检测到单个DSB而触发的。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Dmc 1进行的重组事件。 第二，将评估已知引起有丝分裂细胞周期延迟以响应DNA损伤的突变（RAD 9、RAD 17、RAD 53、TEL1）在减数分裂中的HO DSB之后触发SC形成中的作用。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联会，或者HO诱导的SC形成是否发生在非同源染色体之间。最后，正在进行一个实验系统，以确定每个染色体一个HO诱导的DSB是否有效地指导缺乏任何其他交叉事件的染色体上的适当染色体分离。