Conserved and unique features regulating meiotic crossover formation

Magee-Womens Research Institute and Foundation received a $919,914 Project Grant award from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), effective September 9, 2025 through September 8, 2027. The research project investigates conserved and unique features regulating meiotic crossover formation—the exchange of DNA between homologous chromosomes during meiosis. The research addresses a critical gap in understanding how the core machinery components, specifically the double-strand break (DSB) formation machinery in nematodes, regulate the number, timing, and placement of DNA breaks essential for proper chromosome segregation during sexual reproduction. The research employs an integrated approach combining structural modeling, biochemistry, genetics, and comparative evolutionary cell analysis to elucidate protein interactions and regulatory mechanisms underlying meiotic DSB formation.

The deliverables from this award include functional analyses of SPO-11 and DSB-1 proteins and their domain contributions to regulatory aspects of DSB formation, advancing fundamental understanding of biological processes underlying human reproduction and genetic integrity. As a basic biomedical research initiative supported by NIGMS, the project contributes to the broader scientific foundation for advances in disease diagnosis, treatment, and prevention by elucidating molecular mechanisms conserved across eukaryotic organisms.

Abstract

All eukaryotes on the planet evolved the ability to form haploid gametes through the specialized cell division process of meiosis. Central to meiosis is crossover (CO) formation, the exchange of DNA between homologous chromosomes - a process that both increases diversity and ensures proper segregation of the homologs. Ironically, COS initiate with the formation of double-strand breaks (dsbs) considered the most dangerous DNA lesions because MIS- or unrepaired breaks can lead to chromosome aberrations including translocations and inversions, among others. However, if meiotic dsbs are not formed, chromosomes segregate randomly at meiosis, resulting in aneuploid gametes, and subsequent embryonic lethality or birth abnormalities upon fertilization. Thus, meiotic break formation must be tightly regulated to ensure that neither too few nor too many lesions are made. Dsbs are catalyzed by the endonuclease SPO11 in collaboration with accessory factors that regulate number, timing and placement of dsbs. Despite the ubiquity of DSB formation for meiosis, we know very little about how the different components of the core machinery contribute to these regulatory functions, in part because germline genes are among the fastest evolving in our genome. This proposal addresses this critical GAP in our understanding by interrogating the interactions between, and regulation of, the nematode DSB machinery. Our prior studies found that DSB-1 interacts with both SPO-11 and him-5 provides a mechanism to bring the break machinery to the chromosome axis where strand exchange is executed. This grant combines structural modeling, biochemistry, genetics and comparative evolutionary cell approaches in 3 specific aims. In aim 1 we propose to perform functional analyses of the SPO-11 and DSB-1 proteins and the contributions of different domains to the various regulatory aspects of DSB formation. We will test the hypothesis suggested by our preliminary data that SPO-11 autoregulates its own activity. We will also create new tools to interrogate SPO-11 binding partners in vivo. Our preliminary studies have also revealed that the him-5 exhibits a tightly regulated nuclear mobilization that is functionally relevant. We hypothesize that nuclear retention represents a previously unrecognized way to control the proper timing and repair of dsbs. In aim 2, we will follow up on these novel observations and address the functional significance of him-5 sub-cellular localization with DSB-1 and its regulation by DSB signaling. In aim 3, we will deploy knockdown and gene swaps to interrogate the conservation of the DSB molecular machinery between c. Elegans and c. Briggsae, nematodes that have maintained strong synteny and recombination maps despite 100MY of independent evolution. We hypothesize that comparisons between the conserved dsbs factors will shed light both on the core parts of the DSB machinery that maintain tight CO control and on the flexibility of these factor to adapt to evolutionary forces. Overall, this study will reveal fundamental mechanistic insights into the regulation and evolution of meiotic DSB formation.