Human reproductive success and the development of healthy offspring depend on accurate transmission of
genetic material from parent to child. Homologous recombination during meiosis plays a central role in this
genetic transmission by ensuring accurate chromosome segregation. Errors in recombination can lead to
aneuploidy or mutations in gametes that in turn cause miscarriage or developmental defects in children.
Understanding the mechanism and regulation of recombination is thus critical for understanding how meiotic
errors affect human fertility and child development, but the molecular principles of recombination remain
incompletely understood because of a paucity of biochemical and structural information. Meiotic recombination
initiates with DNA double-strand breaks (DSBs) made by the Spo11 protein in collaboration with a suite of
accessory factors. We recently overcame longstanding barriers to progress by purifying for the first time
recombinant complexes of DSB-promoting proteins. Building on this advance, the Keeney and Patel labs
propose to extend their ongoing collaboration to combine biochemical, structural, and single molecule
biophysical approaches in vitro with functional experiments in vivo to illuminate the molecular principles that
govern how DSB formation by Spo11 occurs. By conducting these studies in parallel on proteins from mouse
and Saccharomyces cerevisiae, we will dive deeply into the mechanisms of evolutionarily conserved processes
while retaining the ability to explore mammal-specific aspects. Aim 1 will focus on a “core complex” of Spo11
with its direct binding partners TOP6BL (mammals) and Rec102–Rec104–Ski8 (yeast). We will apply cryo-EM,
x-ray crystallography, and computational modeling along with biochemical studies to define the structure of
Spo11 core complexes and their critical protein-protein and protein-DNA interfaces. We will also test the
physiological relevance of our structural and biochemical findings in vivo. To this end, we will use molecular
genetic, genomic, and cytological studies in yeast and will employ a novel approach to parallelized genetic
screening in mouse by competitively transplanting pools of genetically modified spermatogonial stem cells into
testes of germ cell-depleted mice. Aim 2 will focus on the conserved accessory proteins Rec114, Mei4, and
Mer2, which are important as a nexus for regulating DSB timing, number and location. We will use NMR
spectroscopy, x-ray crystallography, cryo-EM, and computational modeling to define the structures and protein-
protein interfaces of heterotrimeric Rec114–Mei4 complexes and of homotetrameric Mer2 complexes. We will
use bulk biochemical and single molecule biophysical approaches to define the mechanism and dynamics
behind the cooperative assembly of these proteins to form nucleoprotein condensates on DNA, which we
hypothesize to be a central feature of their ability to support Spo11 activity. We will also apply a battery of in
vivo assays to test functional predictions arising from the structural and biochemical findings.