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

Spo11 对减数分裂 DNA 双链断裂形成的机制见解：对遗传重组和非整倍性的影响

• 批准号: RGPIN-2014-04070
• 负责人: Masson, JeanYves
• 金额: $ 3.42万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566275
• 关键词: Mechanistic; insights; into; meiotic; DNA

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.

在酵母中，减数分裂通过细胞分裂产生单倍体配子或孢子，细胞分裂包括一轮DNA复制，随后是两次细胞分裂。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。这些分裂之前有一个独特的减数分裂前期，在此期间同源染色体突触并进行重组。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，从而产生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，因为同源物之间的交叉无法解决导致染色体分离缺陷，如不分离（染色体不分离）和非整倍体（定义为染色体数目错误的细胞）。酵母中的减数分裂重组是由II型拓扑异构酶Spo11 （S. pombe中的Rec12）产生的双链断裂引发的。Spo11敲除小鼠显示染色体突触缺陷，这表明重组的启动先于减数分裂i，并且是正常染色体突触所必需的。Mre11， Rad50和Nbs1蛋白（MRN）参与Spo11的去除和dsb的加工，以形成重组的3 ' -单链尾DNA。然后，切除的dsb被用来入侵同源双工DNA，这一过程需要RAD51、DMC1、HOP2和MND1基因，从而实现遗传信息的交换（Masson和West， 2001）。这种入侵导致Holliday结的形成，Holliday结被迁移和解决，以完成遗传信息的交换和遗传多样性。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缺乏以及可作为减数分裂重组模型的人类减数分裂细胞系的缺乏的阻碍。为了解决这一问题，我们的目标是利用分裂酵母和秀丽隐杆线虫模型来研究减数分裂重组，因为减数分裂过程在真核生物中得到了很好的保存。虽然遗传学研究已经确定了对Spo11的需求，但对其生化功能知之甚少。其中一个原因是，这种蛋白质的纯化是出了名的困难。我们承担了极其困难的任务，将Spo11提纯到均匀性。我们用一种新的方法纯化了S. pombe的Spo11蛋白，发现该蛋白具有诱导超螺旋DNA双链断裂的活性。对于这一更新，我们将以这些突破性的进展为基础，从功能上剖析Spo11同源物是如何从DNA双链断裂中产生和去除的。我们的目标是：(1)纯化Spo11并广泛表征其体外DNA切割和去除机制。我们将确定其在减数分裂过程中活性所必需的辅助因子。(2)Spo11突变体或相互作用体将用于监测这些蛋白在pombe和秀丽隐杆线虫染色体分离和减数分裂中的作用。在短期内，本研究将丰富我们对减数分裂重组的认识，并在理解重组与染色体分离之间的关系方面迈出一步。从长远来看，这些研究的结果对于理解导致出生缺陷的减数分裂缺陷以及21三体是非常重要的。