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 由细胞监控。In S.在酿酒酵母中，重组由DNA启动 Spo 11蛋白催化的双链断裂（DSB）。我们最近克隆了 并破坏小鼠Spo 11。我们的研究结果强烈表明， 重组起始高度保守。我们建议继续使用 Spo 11基因敲除小鼠研究减数分裂重组和细胞反应 重组缺陷。具体目标如下： 1.检查点对重组缺陷的反应。我们提出哺乳动物 具有依赖性和非依赖性DNA损伤的减数分裂 检查站为了验证这个想法，我们将确定上位关系， 早期（Spo 11-/-）、中期（Dmc 1-/-和Msh 5-/-）和晚期（Mlh 1-/-）之间 重组突变体，其引起不同的检查点应答。我们还将 确定p53是否在重组引起的卵母细胞凋亡中起作用 缺陷 2. 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， 被认为参与修复减数分裂DSB。我们将确定基因 通过分析Spo 11-/-和其他 变种人此外，我们还开发了一个系统， 使用稀有切割核酸内切酶I-Scel在精母细胞中的DSB。我们将 确定这种断裂在野生型减数分裂过程中是如何修复的， 将该系统引入Spo 11-/-后台， 推定的重组蛋白集中在确定的DSB位点上。 3. spo 11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表达载体的控制下表达Spo 11转基因。 启动子，以确定已知Spo 11剪接的功能意义 变体，并将表达催化失活突变体，以确定Spo 11 在减数分裂期间具有不依赖于DSB的作用。我们也会用这些转基因 以滴定Spo 11在体内的活性。这将使我们能够确定细胞如何 对适度重组缺陷的反应，而不是灾难性的 重组缺陷无效突变体中的缺陷。 4.减数分裂重组与卵母细胞纺锤体之间的相互作用。交叉 形成被认为是第一次减数分裂的正确组装的关键 小鼠卵母细胞纺锤体。我们将进一步探讨这种关系， 检测培养的Spo 11-/-卵母细胞中纺锤体的形成。我们还将 明确测试Spo 11是否是减数分裂交换所必需的， 突变卵母细胞交叉形成。

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.