Role of Spo11 and recombination in mouse meiosis

描述(由申请人提供)：我们的目标是了解 哺乳动物减数分裂重组，并确定这一过程是如何进行的 由细胞监控。在酿酒酵母中，重组是由dna启动的。 Spo11蛋白催化的双链断裂(DSB)。我们最近克隆了 并扰乱了老鼠Spo11。我们的结果有力地表明， 复合引发是高度保守的。我们建议继续使用 Spo11基因敲除小鼠研究减数分裂重组及其细胞反应 到复合缺陷。具体目标如下： 1.对重组缺陷的检查点响应。我们已经提出哺乳动物 有性二态DNA损伤依赖和独立的减数分裂 检查站。为了测试这个想法，我们将确定上位关系 介于早期(Spo11-/-)、中期(Dmc1-/-和Msh5-/-)和晚期(MLH1-/-)之间 重组突变体，可引起不同的检查点反应。我们还将 确定P53在重组引起的卵母细胞凋亡中是否起作用 缺陷。 2.推测的DSB修复蛋白及其在哺乳动物减数分裂中的作用 重组。许多蛋白质在减数分裂染色体上形成焦点，因此 推测与修复减数分裂双链断裂有关。我们将确定基因 通过分析Spo11-/-和其他因素对这些焦点的形成要求 变种人。此外，我们还开发了一个系统来生成特定于站点的 用稀有内切酶I-SCEL检测精母细胞中的DSB。我们会 确定这样的断裂在野生型减数分裂过程中是如何修复的，并将 将此系统引入Spo11-/-后台以监控 推测重组蛋白集中在一个确定的DSB位点上。 3.Spo11功能和细胞对部分重组缺陷的反应。我们 将在精母细胞特异性控制下表达Spo11转基因 启动子用于确定已知Spo11剪接的功能意义 并将表达催化失活突变体以确定Spo11 在减数分裂过程中具有不依赖于双链断裂的作用(S)。我们还将利用这些转基因 体内滴定Spo11活性。这将使我们能够确定细胞如何 应对适度的重组缺陷，而不是灾难性的 重组缺陷零突变体中的缺陷。 4.减数分裂重组与卵母细胞纺锤体的相互作用。交叉 形成被认为是正确组装第一次减数分裂的关键。 小鼠卵母细胞中的纺锤体。我们将通过以下方式进一步探讨这种关系 检测培养的Spo11-/-卵母细胞纺锤体的形成。我们还将 通过分析来明确测试减数分裂交换是否需要Spo11 突变卵母细胞的交叉形成。

DESCRIPTION (provided by applicant): Our goal is to understand the mechanism of meiotic recombination in mammals, and to determine how this process is monitored by the cell. In S. cerevisiae, recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. We recently cloned and disrupted mouse Spo11. Our results strongly suggest that the mechanism of recombination initiation is highly conserved. We propose to continue using the Spo11 knockout mouse to study meiotic recombination and the cellular responses to recombination defects. The Specific Aims are as follows: 1. Checkpoint responses to recombination defects. We have proposed that mammals have sexually dimorphic DNA damage-dependent and -independent meiotic checkpoints. To test this idea, we will determine the epistasis relationships between early (Spo11-/-), middle (Dmc1-/- and Msh5-/-), and late (Mlh1-/-) recombination mutants, which elicit distinct checkpoint responses. We will also determine if p53 plays a role in oocyte apoptosis caused by recombination defects. 2. Putative DSB repair proteins and their role in mammalian meiotic recombination. Many proteins form foci on meiotic chromosomes and thus are assumed to be involved in repairing meiotic DSBs. We will determine the genetic requirements for formation of these foci by analyzing Spo11-/- and other mutants. In addition, we have developed a system to generate a site-specific DSB in spermatocytes using the rare-cutting endonuclease I-Scel. We will determine how such a break is repaired during wild-type meiosis and will introduce this system into a Spo11-/- background to monitor the assembly of putative recombination protein foci on a defined DSB site. 3. Spo11 function and cellular responses to partial recombination defects. We will express Spo11 transgenes under the control of a spermatocyte-specific promoter to determine the functional significance of known Spo11 splice variants and will express catalytically inactive mutants to determine if Spo11 has a DSB-independent role(s) during meiosis. We will also use these transgenes to titrate Spo11 activity in vivo. This will allow us to determine how cells respond to a modest recombination defect, as opposed to the catastrophic defects in recombination-defective null mutants. 4. Interplay between meiotic recombination and the oocyte spindle. Chiasma formation is thought to be critical for proper assembly of the first meiotic spindle in mouse oocytes. We will further explore this relationship by examining spindle formation in cultured Spo11-/- oocytes. We will also explicitly test whether Spo11 is required for meiotic crossing over by assaying chiasma formation in mutant oocytes.