Project Summary
Meiosis couples one round of DNA replication, high-frequency recombination between homologs, and two
rounds of chromosome segregation to produce haploid meiotic products. Meiotic recombination is required for
the proper segregation of homologs in meiosis, and it generates genetic diversity required for the process of
natural selection. Interestingly, meiotic recombination is clustered at “hotspots” that regulate its frequency
distribution throughout the genome. Our model system, fission yeast, led to the discovery that discrete DNA
sequence motifs and their binding proteins position the initiation of recombination at hotspots. They do so by
inducing histone PTMs and nucleosome displacement, which promote locally the catalysis of recombination-
initiating dsDNA breaks by the basal recombination machinery (Spo11/Rec12 complex). The general, DNA site-
dependent mechanisms are conserved between species that diverged about 400 million years ago and are
implicated by association to be even more broadly conserved. Remarkably, a screen of short, randomized DNA
sequences generated by base-pair substitutions in the fission yeast genome—which directly analyzed rates of
meiotic recombination in about 46,000 independent clones—identified 202 distinct, short DNA sequence
elements that activate recombination hotspots. These striking findings suggest the most meiotic recombination,
like most transcription, is positioned and regulated by discrete DNA sites and their binding proteins. However,
only five of the 202 hotspot-activating DNA sequences have been defined functionally at single-nucleotide
resolution, and the binding/activator proteins have only been identified for three of the DNA sites. Our long-term
goal is to define systematically the discrete DNA sites and binding/activator protein codes of meiotic
recombination. First, we will use a newly developed tool called “targeted forward genetics” (TFG), which can
generate more than 100,000 independent allele replacements in a single experiment, to define at single-
nucleotide resolution the discrete DNA sequence motifs required for hotspot activity in vivo. Second, we will use
an approach called “DNA affinity capture with mass spectrometry” (DAC-MS), coupled with a tandem mass
tagging (TMT), triple-stage mass spectrometry (MS3) strategy that can analyze many samples simultaneously,
to identify the candidate binding/activator proteins. Candidates will be validated for DNA site-specific binding
and hotspot activation in vivo. Third, we shall test the hypothesis that the different cis-acting regulatory modules
each promote recombination via a common downstream mechanism that involves chromatin remodeling. This
systematic, multifaceted approach will provide new insight into the mechanisms (and discrete codes) that
position meiotic recombination, which has implications for the etiology of meiotic aneuploidies (e.g., Down's
syndrome), for linkage mapping, and for the evolutionary dynamics of genomes.