Meiotic crossovers (CO) are critical for the formation of chiasmata, which in turn ensure the balanced segregation of homologous chromosomes at meiosis I. COs are produced by homologous recombination (HR), which is triggered at meiosis by the formation of numerous programmed DNA double-strand breaks (DSBs). Most species make very few COs per chromosome pair (in the range of 1 to 3) despite a substantial excess of DSBs. C. elegans stands at one end of this spectrum, usually forming just one CO per chromosome pair. Thus, HR is tightly regulated during C. elegans meiosis to ensure that one, and only one, CO is formed during prophase I and that genomic integrity is restored prior to cell division. We are using direct genetic screening approaches to elucidate and decipher the mechanisms underlying meiotic CO formation and its regulation.First, we will report on our analysis of a mutant isolated in a screen for altered numbers of GFP::COSA-1 foci, which mark the sites of COs in late meiotic prophase. Whereas late pachytene nuclei in wild-type worms have six bright GFP::COSA-1 foci, corresponding to the single CO site on each homolog pair, late pachytene nuclei in the
me102 mutant display a single GFP::COSA-1 aggregate, indicative of impaired CO formation. Further, RAD-51 foci corresponding to DSB-dependent HR intermediates are lost in the
me102 mutant, and diakinesis nuclei exhibit frayed, aggregated and/or fragmented chromosomes. Together, these phenotypes are reminiscent of defects observed in
com-1(
t1626) and
mre-11(
iow1) mutants. COM-1 and MRE-11 are part of the conserved MRN/X complex (MRE11, RAD50, and NBS1 in mammals or XRS2 in yeast) and have been implicated in resection of DNA upon DSB formation to promote HR-mediated repair during meiosis. We are currently testing the hypothesis that the
me102 mutation disrupts the previously unidentified C. elegans ortholog of NBS1/XRS2.Secondly, we are investigating mechanisms that limit CO formation by screening for suppressors of a temperature-sensitive
him-14 mutation affecting the conserved MSH-4/MSH-5 complex, which collaborates with COSA-1 to promote meiotic COs. The premise of this screen is that suppressor mutations that improve meiotic chromosome segregation in the
him-14(ts) mutant may identify factors that normally function in antagonizing CO formation.