The role of the Drosophila meiotic MCM proteins in crossover formation. Kathryn P. Kohl, Corbin D. Jones, Jeff Sekelsky. University of North Carolina at Chapel Hill, Chapel Hill, NC.

   Meiotic recombination increases genetic diversity and aids the proper segregation of homologous chromosomes through the formation of crossovers (COs). To prevent aberrant crossing over the formation of COs is highly regulated. One method of regulation is through the use of anti-CO helicases, which unwind inappropriate recombination intermediates, and pro-CO proteins that prevent these helicases from unwinding intermediates destined to form COs. The Msh4-Msh5 complex has been shown to antagonize the anti-CO helicase Sgs1, the S. cerevisiae Bloom syndrome helicase BLM ortholog. All metazoan genomes have Msh4 and Msh5 except Drosophila and Glossina morsitans (tsetse fly). We identified a novel complex of mini-chromosome maintenance proteins (mei-MCMs) that functionally replace Msh4-Msh5 in flies (Kohl et al. Science 2012). Two of these proteins, MEI-217 and MEI-218, are encoded on one dicistronic mRNA and are structurally predicted to contain MCM N- and C-terminal domains, respectively. The third complex member, REC, was found to be under strong positive selection prior to the split of Glossina from Drosophila, suggesting natural selection drove the repurposing of REC into an antagonist of DmBLM. COs are nearly absent in mei-MCM mutants, but removal of DmBLM in these mutants restores CO formation, supporting the hypothesis that the mei-MCMs promote crossing over by blocking the anti-CO activities of DmBLM. We are now examining the role of the mei-MCMs in CO interference. We created flies with mutations in RECs Walker A and Walker B ATPase motifs. We found that mutation of the Walker A motif allowed normal chromosome disjunction but increased the number of double and triple COs. The Walker B mutant showed high levels of non-disjunction and very few COs. However, the residual COs were distributed normally - a surprising result since mei-MCM mutants are characterized by an altered CO distribution. We also created a MEI-218 mutant with proper chromosome segregation but increased multiple COs, further suggesting a role for the mei-MCMs in CO interference.