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: 0077257
• 负责人: James Haber
• 金额: $ 38.79万
• 依托单位: Brandeis University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077257&HistoricalAwards=false
• 关键词: Role; Double; Strand; DNA; Break

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.

减数分裂重组在许多重要方面不同于有丝分裂重组。减数分裂重组是由减数分裂特异性Spo 11拓扑异构酶产生的双链断裂（DSB）启动的，需要至少9种其他蛋白质来创造这些断裂发生的染色体环境。目前尚不清楚这些蛋白质是否也决定了Spo 11介导的基因转换的结果，这表明相对于有丝分裂事件，交换水平非常高。减数分裂交换也受到至少两类蛋白质的强烈影响，即作为联会复合体组分的减数分裂特异性Zip蛋白和Mlhl-Msh 4-Msh 5蛋白。相比之下，有丝分裂基因转换可以由位点特异性HO内切核酸酶启动，其不需要其他蛋白质的作用来产生DSB。在该项目中，HO内切核酸酶在减数分裂特异性启动子的控制下表达，因此可以比较减数分裂和有丝分裂中由相同DSB引发的重组事件。这项工作的一个主要目标是使用这个系统来询问是否还需要制造减数分裂DSB所需的其他9种蛋白质来确保高比例的交换。 此外，该系统可用于确定核酸内切酶是否可在染色体III的“冷”区域中诱导高水平的交换，该区域抑制正常的Spo 11诱导的事件。这项工作的第二个主要目标是检查减数分裂交换的控制。这将是一个方面，以评估细胞学手段，如果一个单一的，HO诱导的交换是足以促进在第一次减数分裂染色体均等分离。这种方法解决了这样一个还原分裂是否会发生在SC的情况下，这似乎取决于许多Spo 11介导的DSB的存在下的问题。 第三个目标是使用这种独特的DSB来研究重组的分子机制，其目标是区分减数分裂重组，如HO诱导的有丝分裂重组，是否通过合成依赖性链退火机制进行。 最后，该系统被用来调查的重要问题，一个交叉事件如何干扰交叉在相邻的时间间隔。最近已经确定，一些染色体间隔实际上并不表现出任何显着的干扰酵母。 一旦确定了适当的间隔，将插入一个或两个HO切割位点，以询问HO介导的事件是否干扰相邻的Spo 11诱导的交叉，以及两个HO诱导的交叉是否也显示干扰。