Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.
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. I have recently made important progress by, for the first time, successfully purifying proteins involved in two aspects of meiotic recombination: DSB formation and the final stage of formation of the crossovers that are a central raison-d’être of meiotic recombination. This has opened new avenues for future research that I intend to pursue in my own laboratory. Here, I propose a set of biochemical approaches, complemented by molecular genetics methods, 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, I will 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, I propose to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis. My 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.