Periodic Reporting for period 4 - BrokenGenome (Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.)
Berichtszeitraum: 2024-01-01 bis 2024-06-30
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 the 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 aimed 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 aimed 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 aimed to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis.
Our goal was 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 could be relevant to the development and avoidance of disease states.
The BrokenGenome project aimed 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 aimed 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 aimed to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis.
Our goal was 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 could be relevant to the development and avoidance of disease states.
At the start of this project, the PI (Corentin Claeys Bouuaert) set up his laboratory at the Host Institution (UCLouvain, Belgium) and recruited a team of researchers to work on the four aims of the proposal.
(1) To gain insight into meiotic DSB formation, we developed for the first time a biochemical system that recapitulates the DNA cleavage activity of Spo11. We also investigated the DNA-binding properties of the Spo11 core complex and performed structure-function analyses. Finally, we characterized the structure and function of RMM proteins and their role in the assembly of the DSB machinery by DNA-driven condensation.
(2) We investigated the biochemical properties of meiotic axis proteins Hop1 and Red1 and their relationships with the meiotic DSB proteins. In addition, we characterized the interactions between the DSB protein Mer2 and the histone H3K4me3 reader, Spp1.
(3) To investigate the mechanism of crossover formation, we have made progress in developing an in vitro system using purified crossover-promoting proteins and a novel DNA substrate that mimics a key recombination intermediate, the double Holliday Junction.
(4) Finally, we set up new mass-spectrometry based assays to identify proteins associated with meiotic recombination.
Some of the results from the BrokenGenome project have already been published and presented at international conferences. Currently, the team is finalizing a number of publications describing the bulk of the achievement of the project. Together, the BrokenGenome project has brought important new insights into the molecular processes involved in the formation and repair of DNA double-strand breaks during meiosis.
(1) To gain insight into meiotic DSB formation, we developed for the first time a biochemical system that recapitulates the DNA cleavage activity of Spo11. We also investigated the DNA-binding properties of the Spo11 core complex and performed structure-function analyses. Finally, we characterized the structure and function of RMM proteins and their role in the assembly of the DSB machinery by DNA-driven condensation.
(2) We investigated the biochemical properties of meiotic axis proteins Hop1 and Red1 and their relationships with the meiotic DSB proteins. In addition, we characterized the interactions between the DSB protein Mer2 and the histone H3K4me3 reader, Spp1.
(3) To investigate the mechanism of crossover formation, we have made progress in developing an in vitro system using purified crossover-promoting proteins and a novel DNA substrate that mimics a key recombination intermediate, the double Holliday Junction.
(4) Finally, we set up new mass-spectrometry based assays to identify proteins associated with meiotic recombination.
Some of the results from the BrokenGenome project have already been published and presented at international conferences. Currently, the team is finalizing a number of publications describing the bulk of the achievement of the project. Together, the BrokenGenome project has brought important new insights into the molecular processes involved in the formation and repair of DNA double-strand breaks during meiosis.
The BrokenGenome led to the following key advances:
1) The in vitro reconstitution of the catalytic activity of mouse SPO11 using recombinant proteins. This key achievement defines the minimal machinery required for DNA cleavage and reveals unanticipated insights into the activity of SPO11. From this work emerges a new understanding the the cleavage activity of SPO11 is caused by a weak dimer interface, which makes SPO11 exquisitely dependent on its accessory proteins in vivo.
2) Characterization of the structure and activities of the yeast Spo11 core complex. Our work revealed new insights into the structural organization of the Spo11 complex, its interaction with DNA, the role of Spo11 accessory partners, and the relationship between the poorly-conserved dimer interface of Spo11 and DNA cleavage.
3) Characterization of the structure and activities of the RMM proteins. Our work revealed that RMM proteins assemble the DSB machinery by a mechanism of DNA-dependent biomolecular condensation, defined the molecular organization of RMM proteins, their evolutionary conservation, the role of phosphorylation in RMM condensation, and the interactions between RMM proteins and other meiotic DSB proteins.
4) Relationship between the meiotic chromosome axis and DSB proteins. Our work revealed new interactions between components of the meiotic chromosome axis and RMM proteins, and establishes how these interactions drive the assembly of the meiotic DSB machinery by nucleating RMM condensates.
5) The connection between DSB proteins and chromatin loops. Our work reveals structural insights into the interaction between Mer2 and the H3K4 trimethylation reader, Spp1 and the relationship between this interaction and the condensation activity of Mer2.
6) Development of new methodologies to investigate the mechanism of crossover formation.
1) The in vitro reconstitution of the catalytic activity of mouse SPO11 using recombinant proteins. This key achievement defines the minimal machinery required for DNA cleavage and reveals unanticipated insights into the activity of SPO11. From this work emerges a new understanding the the cleavage activity of SPO11 is caused by a weak dimer interface, which makes SPO11 exquisitely dependent on its accessory proteins in vivo.
2) Characterization of the structure and activities of the yeast Spo11 core complex. Our work revealed new insights into the structural organization of the Spo11 complex, its interaction with DNA, the role of Spo11 accessory partners, and the relationship between the poorly-conserved dimer interface of Spo11 and DNA cleavage.
3) Characterization of the structure and activities of the RMM proteins. Our work revealed that RMM proteins assemble the DSB machinery by a mechanism of DNA-dependent biomolecular condensation, defined the molecular organization of RMM proteins, their evolutionary conservation, the role of phosphorylation in RMM condensation, and the interactions between RMM proteins and other meiotic DSB proteins.
4) Relationship between the meiotic chromosome axis and DSB proteins. Our work revealed new interactions between components of the meiotic chromosome axis and RMM proteins, and establishes how these interactions drive the assembly of the meiotic DSB machinery by nucleating RMM condensates.
5) The connection between DSB proteins and chromatin loops. Our work reveals structural insights into the interaction between Mer2 and the H3K4 trimethylation reader, Spp1 and the relationship between this interaction and the condensation activity of Mer2.
6) Development of new methodologies to investigate the mechanism of crossover formation.