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

点击翻译按钮获取中文摘要

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.