Periodic Reporting for period 2 - DSBSunrise (Identifying the steps required for meiotic DNA double-strand break formation)
Reporting period: 2022-06-01 to 2023-11-30
The DSBSunrise project is investigating several molecular events required for the transfer of genetic material from one generation to the next during sexual reproduction. This is an evolutionarily conserved process. It occurs in most sexually reproducing species. The molecular events studied in this project take place during meiosis, the specialised cell division that ensures the formation of haploid gametes from diploid precursor germ cells.
During meiosis, two divisions occur to halve the chromosome content, and the first, the reductional division, is unique to meiotic cells as it ensures the segregation of the parental (maternal and paternal) homologous chromosomes. For accurate reductional segregation, the maternal and paternal homologues must recognise each other and be topologically linked. The links are established by reciprocal exchanges of DNA through homologous recombination between homologs.
Thus, meiotic cells induce a high frequency of homologous recombination events, such that each pair of homologs undergoes at least one reciprocal exchange or crossover. Homologous recombination during meiosis is a multistep process initiated by the formation of DNA double-strand breaks, followed by their repair using the homologous chromosome as a template.
Collectively, these molecular processes not only ensure the proper transmission of chromosomes to the next generation, but also determine genetic linkage and have long-term consequences for genome evolution by generating novel combinations of alleles, thus contributing to an increase in genetic diversity. Of note, somatic cells use homologous recombination to repair various types of DNA damage, but the meiotic cell is unique in that it has a machinery and associated processes to induce intentional DNA breaks in its genome. Several hundred DNA double-strand breaks are generated in each mouse or human meiotic cell. Tight control is required to ensure that each of these DNA breaks is repaired faithfully by homologous recombination, without errors that could lead to the transmission of genomic changes to the progeny.
The DSBSunrise project aims to investigate several features of DNA break formation in meiotic cells and how they are regulated. It will search for molecular components, address how DNA break sites are organised along chromosomes, how they interact with structural components of the repair mechanism and chromosome organisation, and provide fundamental insights into the complex process of meiotic chromosomal recombination.
During meiosis, two divisions occur to halve the chromosome content, and the first, the reductional division, is unique to meiotic cells as it ensures the segregation of the parental (maternal and paternal) homologous chromosomes. For accurate reductional segregation, the maternal and paternal homologues must recognise each other and be topologically linked. The links are established by reciprocal exchanges of DNA through homologous recombination between homologs.
Thus, meiotic cells induce a high frequency of homologous recombination events, such that each pair of homologs undergoes at least one reciprocal exchange or crossover. Homologous recombination during meiosis is a multistep process initiated by the formation of DNA double-strand breaks, followed by their repair using the homologous chromosome as a template.
Collectively, these molecular processes not only ensure the proper transmission of chromosomes to the next generation, but also determine genetic linkage and have long-term consequences for genome evolution by generating novel combinations of alleles, thus contributing to an increase in genetic diversity. Of note, somatic cells use homologous recombination to repair various types of DNA damage, but the meiotic cell is unique in that it has a machinery and associated processes to induce intentional DNA breaks in its genome. Several hundred DNA double-strand breaks are generated in each mouse or human meiotic cell. Tight control is required to ensure that each of these DNA breaks is repaired faithfully by homologous recombination, without errors that could lead to the transmission of genomic changes to the progeny.
The DSBSunrise project aims to investigate several features of DNA break formation in meiotic cells and how they are regulated. It will search for molecular components, address how DNA break sites are organised along chromosomes, how they interact with structural components of the repair mechanism and chromosome organisation, and provide fundamental insights into the complex process of meiotic chromosomal recombination.
During the first part of the project, we set up several tools to develop our experimental programme. In particular, to monitor the binding of different proteins to the chromatin of meiotic cells, we have optimised protocols for the enrichment of meiotic cells at the right stage and optimised protocols for the detection of the proteins of interest. We developed methods to monitor signal-specificity by performing ad hoc negative controls. This has allowed us to discover novel features of the interactions between proteins involved in recombination and chromosome organisation. We have also optimised bioinformatics tools to analyse the signal from DMC1 ChIP experiments. To gain insight into the proteins directly involved in DSB formation, we identified a direct interaction between TOPOVIBL and REC114, allowing us to propose a model for the catalytic complex. In addition, we used structural analysis to determine the properties of the interactions between the DSB-associated proteins REC114, MEI4 and IHO1.
Our discovery of novel protein-chromatin interactions involving recombination and structural proteins of the chromosome axes provides a basis for predicting models of meiotic chromosome organisation.
Our identification of direct interactions between several DSB proteins provides predictions for the composition of the putative complex that catalyses DNA breaks during meiosis.
Our identification of direct interactions between several DSB proteins provides predictions for the composition of the putative complex that catalyses DNA breaks during meiosis.