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Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.

Periodic Reporting for period 2 - BrokenGenome (Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.)

Reporting period: 2021-01-01 to 2022-06-30

Sexual reproduction depends on the programmed induction of DNA double-strand breaks (DSBs) and their ensuing repair by homologous recombination. This complex process is essential for sexual reproduction because it ultimately allows the pairing and separation of homologous chromosomes during formation of haploid gametes. Although meiotic recombination has been investigated for decades, many of the underlying molecular processes remain unclear, largely due to the lack of biochemical studies.

The BrokenGenome project aims to gain insights into four central problems: (i) How meiotic proteins collaborate to induce DSBs; (ii) How DSB proteins interact with components that form the axes of meiotic chromosomes; (iii) How proteins involved at later stages of recombination form crossovers; and (iv) How crossover proteins interact with components of synapsed chromosomes. For each problem, we aim to set up in vitro systems to probe the activities of the players involved, their interactions with DNA, and their assembly into macromolecular complexes. In addition, we aim to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis.

Our goal is to gain insights into the mechanisms that govern meiotic recombination. Importantly, these mechanisms are intimately linked not only to gamete formation, but also to the general recombination pathways that all cells use to maintain genome stability. In both contexts, our findings will be relevant to the development and avoidance of disease states.
During the first period of this project, the PI (Corentin Claeys Bouuaert) set up his laboratory at the Host Institution (UCLouvain, Belgium), recruited a team of researchers and initiated work on all four aims of the proposal.

(1) To gain insight into meiotic DSB formation, we are developing distinct approaches to reconstitute the catalytic activity of Spo11 based on direct detection of covalent protein-DNA intermediates and site-specific targeting of Spo11 activity; we are investigating the DNA-binding properties of the Spo11 core complex and performing structure-function analyses; and we are characterizing the RMM proteins and their role in the assembly of the DSB machinery by DNA-driven condensation.

(2) We are investigating the biochemical properties of meiotic axis proteins Hop1 and Red1 and their relationships with the meiotic DSB proteins, and set up in vitro systems to investigate interactions between DSB proteins and chromatin using purified nucleosomes and the histone H3K4me3 reader, Spp1.

(3) To investigate the mechanism of crossover formation, we are developing in vitro systems using purified crossover-promoting proteins and a novel DNA substrate that mimics a relevant structure, the double Holliday Junction.

(4) We are developing a system to label recombination-associated nascent DNA in yeast to pull down and identify associated proteins by mass spectrometry.

In sum, the BrokenGenome is well under way and is poised to shed new light onto the molecular processes that break and repair DNA during meiosis.
By the end of the project, we expect to reveal fundamental new insights into the mechanism of meiotic DNA double-strand break formation (Aim 1) and its relationship with chromosome organization (Aim 2), to decipher the mechanism of crossover formation (Aim 3), and to identify new proteins involved in meiotic recombination (Aim 4).
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