Mechanistic insights into meiotic DNA double-strand break formation by Spo11: impact on genetic recombination and aneuploidy

在酵母中，减数分裂通过细胞分裂产生单倍体配子或孢子，细胞分裂由一轮DNA复制和两次细胞分裂组成。第一次分裂或减数分裂I涉及同源染色体彼此分离，而减数分裂II涉及姐妹染色单体的分离，因此类似于有丝分裂。这些分裂之前是一个独特的减数分裂前期，在此期间同源染色体联会和重组。在这个阶段，来自每个亲本的染色体以比营养细胞高100-1000倍的频率重组，导致具有不同遗传特征的配子（Masson和West，2001）。减数分裂重组的结果是基因重排，遗传多样性和适当的染色体分离。这是特别重要的，因为不能解决同源物之间的交叉导致染色体分离缺陷，例如不分离（染色体不分离）和非整倍性（定义为具有错误数目的染色体的细胞）。酵母中的减数分裂重组是由Spo 11（S. pombe），II型拓扑异构酶。Spo 11基因敲除小鼠显示染色体突触缺陷，表明重组的启动先于减数分裂I期间的正常染色体突触，并且是减数分裂I期间正常染色体突触所必需的。Mre 11、Rad 50和Nbs 1蛋白（MRN）参与Spo 11的去除和DSB的加工，以形成重组的3 '-单链加尾DNA。然后，切除的DSB用于侵入同源双链体DNA，该过程需要RAD 51、DMC 1、HOP 2和MND 1基因，导致遗传信息的交换（Masson和West，2001）。这种入侵导致了霍利迪连接的形成，它被迁移和解决，以完成遗传信息和遗传多样性的交换。到目前为止，人类减数分裂重组的生物化学和遗传学研究一直受到人类卵巢或睾丸的不可用性以及缺乏可用作减数分裂重组模型的减数分裂人类细胞系的阻碍。为了解决这个问题，我们的目标是使用裂变酵母和C。由于减数分裂过程在真核生物中是很保守的，因此线虫是研究减数分裂重组的模型。虽然遗传学研究已经确定了Spo 11的需求，但对其生化功能知之甚少。其中一个原因是，众所周知，纯化这种蛋白质非常困难。我们承担了将Spo 11纯化至均一的极其困难的任务。采用新的方法纯化了S. pombe Spo 11中表达的蛋白质，发现该蛋白质具有诱导超螺旋DNA中DNA双链断裂的活性。对于这次更新，我们将在这些突破性进展的基础上，从功能上剖析Spo 11直系同源物如何创建和从DNA双链断裂中移除。我们的目的是：（1）纯化Spo 11，并对其体外切割和去除DNA的机制进行深入研究。（2）Spo 11的突变体或互作子将被用来监测这些蛋白质在S. pombe和C.优雅从短期来看，该研究将丰富我们对减数分裂重组的认识，并对重组与染色体分离之间的关系的理解迈出一步。从长远来看，这些研究的结果将是非常重要的了解减数分裂缺陷导致出生缺陷以及21三体。

Meiosis generates haploid gametes or spores in yeast through a cell division that consists of one round of DNA replication followed by two cell divisions. The first division or meiosis I involves the segregation of homologous chromosomes from each other whereas meiosis II involves the segregation of sister chromatids and is therefore analogous to a mitotic division. These divisions are preceded by a unique meiotic prophase during which homologous chromosomes synapse and undergo recombination. At this stage, chromosomes from each parent recombine at frequencies that are 100-1000 fold higher than in vegetative cells leading to gametes with different genetic signatures (Masson and West, 2001). The consequences of meiotic recombination are gene rearrangements, genetic diversity and proper chromosome segregation. This is of particular importance as failure to resolve chiasmata between homologs results in chromosome segregation defects such as non-disjunction (non-separation of chromosomes) and aneuploidy (defined as a cell having a wrong number of chromosomes). Meiotic recombination in yeast is initiated by the creation of double- strand breaks by Spo11 (Rec12 in S. pombe), a type II topoisomerase. Spo11 knock-out mice display chromosome synapsis defects suggesting that the initiation of recombination precedes and is required for normal chromosome synapsis during meiosis I. The Mre11, Rad50 and Nbs1 proteins (MRN) are involved in the removal of Spo11 and processing of DSBs to form recombinogenic 3’-single-stranded tailed DNA. The resected DSBs are then used to invade homologous duplex DNA, a process that requires the RAD51, DMC1, HOP2 and MND1 genes leading to the exchange of genetic information (Masson and West, 2001). This invasion leads to the formation of Holliday junction, which is migrated and resolved to complete the exchange of genetic information and genetic diversity. So far, biochemical and genetic studies of human meiotic recombination has been hampered by the unavailability of human ovaries or testis and also the absence of a meiotic human cell line that can be used as a model for meiotic recombination. To tackle this problem we aim to use fission yeast and C. elegans models to study meiotic recombination since the process of meiosis is well conserved through eukaryotes. Although genetic studies have established the requirement for Spo11, only little is known on its biochemical function. One reason, is that it is notoriously difficult to purify this protein. We undertook the extremely difficult task of purifying Spo11 to homogeneity. Using a new protocol, we purified S. pombe Spo11 and the protein was found to be active to induce DNA double-strand breaks in supercoiled DNA. For this renewal, we will build on these groundbreaking advances and functionally dissect how Spo11 orthologs creates and are removed from DNA double-strand breaks. Our objectives are: (1) to purify Spo11 and characterize extensively its mechanism of DNA cleavage and removal in vitro. We will identify cofactors necessary for its activity during meiosis and (2) Mutants in Spo11 or interactors will be used to monitor the effect of these proteins in chromosome segregation and meiosis in S. pombe and C. elegans. In short term, the proposed research will enrich our knowledge on meiotic recombination and make a step towards the understanding of the relationship between recombination and chromosome segregation. In long term, the outcome of these studies will be very important to understand meiotic defects leading to birth defects as well as trisomy 21.