Meiotic recombination performs two key functions: 1) to promote genetic diversity by reassorting traits and 2) to establish temporary attachments between pairs of homologous chromosomes (bivalents) necessary for their future segregation. Recombination is initiated by the introduction of DNA double-strand breaks (DSBs). Some DSBs are repaired to form crossovers (COs) between homolog pairs, and the rest are repaired as noncrossover products to restore genome integrity. Although DSBs are required for CO formation, they may lead to genomic instability if they are not repaired or are repaired erroneously. Thus, meiotic cells use surveillance mechanisms to ensure that enough DSBs are created to guarantee a CO on each homolog pair while limiting excess DSBs that may endanger the genome. Without appropriate DSB formation and repair, COs fail to form and univalents (unattached homologs) are observed at late meiotic prophase, leading to chromosome missegregation during the meiotic divisions and aneuploidy in resulting progeny. Here I have identified a new component of the meiotic recombination machinery defined by the
me6ts mutation.
me6ts was previously isolated in a genetic screen as a temperature-sensitive mutant with elevated chromosome missegregation and univalents at the nonpermissive temperature. Based on several lines of evidence, I found that the primary defect in
me6ts is in DSB formation. Nuclear localization of DSB-2 (a chromatin-associated protein required for efficient DSB formation) is greatly reduced in early meiotic prophase nuclei the
me6ts mutant. Further, cytological foci of RAD-51 (a DNA strand exchange protein that marks the sites of processed DSBs) are greatly reduced in number. Finally, introducing DSBs through irradiation is sufficient to restore CO formation. Complementation tests and mapping experiments suggest that the
me6ts mutation is not within any currently known meiotic gene. Thus, I have used genomic sequencing of a backcrossed
me6ts strain and have developed a sequence-analysis pipeline to identify homozygous candidate mutations. I am currently in the process of testing candidate mutations for
me6ts-phenotype causality by RNAi, CRISPR/Cas9-mediated knock-outs, and complementation assays. My future experiments aim to identify how the product of the gene defined by
me6ts facilitates DSB formation through cytological examination of DSB-associated proteins and their regulators in
me6ts mutants.