Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. This study tests the hypothesis that DSB control involves a network of intersecting negative regulatory circuits. Using multiple complementary methods, we show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. ZMM-dependent DSB control is genetically distinct from a pathway tying break formation to meiotic progression through the Ndt80 transcription factor. These counterintuitive findings suggest that homologous chromosomes that have successfully engaged one another stop making breaks. Genome-wide DSB maps uncover distinct responses by different subchromosomal domains to the zmm mutation zip3, and show that Zip3 is required for the previously unexplained tendency of DSB density to vary with chromosome size. Thus, feedback tied to ZMM function contributes in unexpected ways to spatial patterning of recombination.

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.

The budding yeast Saccharomyces cerevisiae has proven to be a rich source of information about the mechanisms and regulation of homologous recombination during meiosis. A common technique for studying this process involves microdissecting the four products (ascospores) of a single meiosis and analyzing the configuration of genetic markers in the spores that are viable. Although this type of analysis is powerful, it can be laborious and time-consuming to characterize the large numbers of meioses needed to generate statistically robust data sets. Moreover, the reliance on viable (euploid) spores has the potential to introduce selection bias, especially when analyzing mutants with elevated frequencies of meiotic chromosome missegregation. To overcome these limitations, we developed a versatile, portable set of reporter constructs that drive fluorescent protein expression specifically in only those spores that inherit the reporter. These spore-autonomous fluorescence constructs allow direct visualization of inheritance patterns in intact tetrads, eliminating the need for microdissection and permitting meiotic segregation patterns to be ascertained even in aneuploid spores. As proof of principle, we demonstrate how different arrangements of reporters can be used to quantify crossover frequency, crossover interference, gene conversion, crossover/noncrossover ratios, and chromosome missegregation.