Final Report Summary - DNA-REPAIR-CHROMATIN (Biochemical reconstitution of DNA repair reactions on physiological chromatin substrates)
Research funded by the ERC Consolidator Award "DNA-REPAIR-CHROMATIN 311336" has been focussed on understanding the molecular and genetic control of steps within the genetic recombination process that takes place within meiotic cells. This biological process is broadly conserved across all sexually reproducing species and enables the production of genetically unique haploid gametes suitable for fertilisation—thereby generating the genetic diversity upon which natural selection may act. Within this programme we were focussed on characterising the main steps of the recombination reaction: DNA break formation by Spo11, Spo11 removal, DNA resection, repair via strand exchange, and resolution into crossover and non-crossover products, and to understand how these sequential steps take place within the complex physiological structure of compacting eukaryotic chromosomes, complete with chromatin structure and higher order levels of organisation. The project was approached with two complementary ideas: exploration of the use of biologically-isolated recombination intermediates (from meiotic cells) as biochemical substrates for repair reactions; paired with more classical use of and development of genetic and molecular biology assays to explore function and regulation. The biochemical reconstitution experiments proved the most challenging, yet culminated in significant insight into the steps of Spo11 removal and resection initiation—especially when paired with complementary genetic and molecular assays. Via collaboration with the Cejka laboratory (Zurich), we determined that phosphorylation of Sae2 regulates the efficiency of Spo11 removal from DNA ends, and that this is likely achieved via preferential interaction with the Mre11 nuclease. We explored mechanism to remove Spo11 in vitro, and utilised the mammalian TDP2 enzyme as a way to clean up DNA ends, but is surprisingly unable to reproduce this role in vivo during meiosis. This work permitted us to explore DNA break formation and the influence of higher order chromatin organisation, by developing methods to map with single nucleotide resolution, and strand-specificity, Spo11 in meiosis, and to begin to compare this to Top2 activity across the genome in both yeast and human cells. By optimising kilobase-resolution HiC maps for meiotic cells, we will be able to correlate these with changes in chromosomes with DNA break formation and repair. Finally, by analysing the genetic control of recombination outcomes, we have explored the regulation and spatial distribution of DNA breaks made by Spo11, and of the resulting crossover and noncrossover products. Both of these latter two projects involved developing international collaborations with bioinformatics teams specialised in data analysis and processing, and in the generation of detailed 3D polymer simulations of meiotic chromosomes. Collectively our research has, and is, furthering our understanding of the complex interplay between components of this essential recombination pathway that takes place in meiosis, and enabling our ability to compare and contrast patterns and relationships to other cell types, cell cycle stages and organisms in order to more broadly understand the biological mechanisms that underpin these processes.