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-2015-04551
• 负责人: Masson, JeanYves
• 金额: $ 2.77万
• 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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687154
• 关键词: Mechanistic; insights; into; meiotic; DNA

Meiosis generates haploid gametes or spores in yeast. In meiotic prophase, 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. 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. 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. 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 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. 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. The outcome of these studies will be very important to understand meiotic defects leading to birth defects as well as trisomy 21. **

在酵母中减数分裂产生单倍体配子或孢子。在减数分裂前期，同源染色体发生联会和重组。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。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通过Spo 11产生双链断裂而启动的（S.Rec 12）。pombe），II型拓扑异构酶。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DNA，这一过程需要RAD 51、DMC 1、HOP 2和MND 1基因，从而导致遗传信息的交换。这种入侵导致了霍利迪结的形成，霍利迪结被迁移和分解，完成了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殖酵母研究减数分裂重组，因为减数分裂的过程是通过真核生物很好地保存。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.粟酒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。这些研究的结果将是非常重要的，以了解减数分裂缺陷导致出生缺陷以及21三体。**