The role of Spo11 in mammalian meiosis

工作总结：染色体双链断裂(DSB)对基因组完整性和细胞生存构成重大和直接的危险。DSB可以通过外源因素(辐射和化学物质)、内源原因(例如，复制叉子倒塌)以及在发育过程中(Ig重组和类别转换，以及减数分裂)的作用而产生。双链断裂可以通过非同源末端连接或同源重组(HR)修复。这两条途径在包括哺乳动物在内的大多数生物体中都同样重要(在Sonoda等人中进行了审查。(2006)DNA修复5,1021))。 HR在所有形式的生命中都是常见的，是一个涉及许多基因产物的多步骤过程。在真核生物中，生物学上最重要的重组形式是减数分裂中同源染色体(同源)之间的遗传信息交换(见Gerton和Hawley(2005)自然修订版Genet)。6,477)。减数分裂重组是真核生物遗传学中的中心现象，它保证了减数分裂第一次分裂时染色体的正确分离(防止不分离)，是决定基因组进化的主要力量。在所有生物中，同源重组与DNA修复和复制密不可分。RAD51和减数分裂特异的Dmc1蛋白都是细菌RecA(典型的同源重组蛋白)的同源蛋白，在包括人和小鼠在内的大多数真核生物中都被发现，纯合子的RAD51-/小鼠ES细胞是不活的。因此，对RecA同源物的研究不仅应该对同源重组有深刻的认识，而且对基因稳定性和细胞增殖的调控也应该有深刻的认识。 在所有生物体的减数分裂中，包括哺乳动物(Romanienko和Camerini-Otero(2000)Mol.细胞6,975)，Spo11，一种II型类似拓扑异构酶，在每个核的许多位置(哺乳动物中有几百个或更多)切割染色体DNA(在Keeney和Neale(2006)Biochem中回顾)。SoC。翻译过来的。34,523)。在哺乳动物中，减数分裂核中的内源DSB比有机体一生中的任何体细胞核中都要多，至少是生物体的100倍。因此，在体细胞中调用的整个HR机制加上额外的减数分裂特异蛋白是有效修复减数分裂中DSB所必需的。这些基因包括P53、BRCA1、BRCA2、RAD51、Dmc1、ATM、ATR、DNA-PKcs、Chk2、Nbs1、Mre11、RPA、BLm等。 Spo11是酿酒酵母减数分裂染色体联会所必需的。令人惊讶的是，Spo11同源物对于线虫和黑腹线虫的突触是必不可少的，但却是减数分裂重组所必需的。我们已经产生了一个SPO11小鼠基因敲除，以研究该基因在哺乳动物中的生物学功能。破坏小鼠SPO11会导致不孕不育。精母细胞在粗线期之前停止，很少或没有同源突触，并经历凋亡(Romanienko和Camerini-Otero(2000)Mol.牢房6,975)。令人惊讶的是，Spo11杂合性挽救了在缺乏ATM(共济失调毛细血管扩张，突变)双链断裂信号蛋白的小鼠中看到的减数分裂前期停滞(Bellani，Romanienko，Casatti和Camerini-Otero(2005)J.Cell Science 118,3233)。 最近，我们用兔抗Spo11蛋白的单抗证实，事实上大多数Spo11蛋白在前期I表达较晚，并在细胞质中发现。此外，我们已经能够证明，α-异构体表达最强烈，表达较晚。 最后，我们重新检查了在小鼠和人类中发现的两种主要剪接异构体Spo11，SPO11α(跳过外显子2)和SPO11β的表达谱。我们的数据表明，SPO11β在引入启动减数分裂重组的中断方面发挥了重要作用。此外，我们发现SPO11α是表达最多的形式，但主要是在引入DSB之后，这种表达在精母细胞和卵母细胞中都可以看到。我们提出了SPO11α在前期中期和晚期的作用，推测其在雄性和雌性小母细胞中都是一种拓扑异构酶。

Summary of work: Chromosomal double-strand breaks (DSBs) pose a significant and immediate danger to genome integrity and cell survival. DSBs can arise through the action of exogenous agents (radiation and chemicals), endogenous causes (e.g., collapsed replication forks) and in the course of developmental programs (Ig recombination and class switching, and meiosis). DSBs are repaired by either nonhomologous end joining or homologous recombination (HR). Both pathways are equally important in most organisms, including mammals (reviewed in Sonoda et al. (2006) DNA Repair 5, 1021)). HR is common to all forms of life and is a multistep process involving many gene products. In eukaryotes, the biologically most important form of recombination is the exchange of genetic information between homologous chromosomes (homologs) in meiosis (reviewed in Gerton and Hawley (2005) Nature Rev. Genet. 6, 477). Meiotic recombination is the central phenomenon in the genetics of eukaryotes, ensures the proper segregation of chromosomes at the first division of meiosis (prevents non-disjunction) and is the main force shaping the evolution of genomes. In all organisms, homologous recombination is inextricably related to DNA repair and replication. Rad51 and the meiosis-specific Dmc1 protein, both homologues of bacterial RecA (the prototypical homologous recombination protein), have been identified in most eukaryotes including man and mouse, and homozygous Rad51 -/- mouse ES cells are not viable. Thus, the study of RecA homologues should yield insights into not only homologous recombination but also the regulation of gene stability and cell proliferation. In meiosis in all organisms, including mammals (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975), Spo11, a type II-like topoisomerase, cleaves the chromosomal DNA at many sites (a couple of hundred or more in mammals) in each and every nucleus (reviewed in Keeney and Neale (2006) Biochem. Soc. Trans. 34, 523). In mammals there are more endogenous DSBs in meiotic nuclei than in any somatic nuclei in the life of an organism, by a factor of at least 100. Hence the entire arsenal of the HR machinery invoked in somatic cells plus additional meiosis-specific proteins are required for the efficient repair of DSBs in meiosis. These include p53, Brca1, Brca2, Rad51, Dmc1, Atm, Atr, DNA-PKcs, Chk2, Nbsl, Mre11, Rpa, Blm, etc. Spo11 is required for meiotic chromosomal synapsis in S. cerevisae. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975). Surprisingly, Spo11 heterozygosity rescues the meiotic prophase arrest seen in mice lacking the Atm (ataxia telangiectasia, mutated ) double-strand break signaling protein (Bellani, Romanienko, Cairatti and Camerini-Otero (2005) J. Cell Science 118, 3233). Most recently, we have used a rabbit monoclonal antibody to the Spo11 protein to confirm that in fact most of the Spo11 protein is expressed late in prophase I and is found in the cytoplasm. Furthermore, we have been able to show that it is the alpha-isoform that is most abudantly expressed and that is expressed late. Finally, we have reexamined the expression profiles of the two major splicing isoforms of Spo11, Spo11alpha (exon 2 skipped) and Spo11beta found in both mice and humans. Our data argues for a major role for Spo11beta in introducing the breaks that initiate meiotic recombination. Furthermore, we find that Spo11alpha is the form that is most expressed but mainly after the DSBs are introduced and this expression is seen in both spermatocytes and oocytes. We propose a role for Spo11alpha in mid- to late prophase, presumably acting as a topoisomerase, in both male and female meiocytes.