| The Drosophila meiotic recombination checkpoint couples the progression of the meiotic program with the developmental patterning of the Drosophila oocyte. Programmed double-stranded DNA breaks are made in order to initiate meiotic recombination. If these breaks are unable to be repaired, the persistence of recombination intermediates activates the meiotic recombination checkpoint. Chronic activation of the meiotic checkpoint results in the failure to properly pattern the dorsal-ventral axis of the eggshell and normally organize the oocyte nuclear DNA.;In order to further understand the meiotic recombination checkpoint, and the related Drosophila DNA damage checkpoints occurring in the somatic tissues, I, in collaboration with other members of the laboratory, performed both an unbiased forward genetic screen and utilized a candidate gene approach. A collection of EMS mutant lines was made and screened for suppressors of the eggshell defect of spnB, a meiotic DNA repair mutant. Twenty-two homozygous viable and 38 homozygous lethal lines that suppress the eggshell defects of spnB mutants were isolated. I mapped one of the suppressing mutations to the general translation factor Eif1A. eif1a mutations dominantly suppress the Gurken translation defects, but not the grk RNA localization or karysome defects, of spnB mutants, arguing for a direct role in checkpoint-activated inhibition of Gurken translation.;I studied two candidate genes, hus1, a gene involved in checkpoint signal transduction in mammals and yeast, and, brca2 , a gene required for homologous recombination in higher organisms. I demonstrated that, in addition to a requirement during the meiotic recombinant checkpoint, hus1 is also required in somatic tissues for the DNA damage response after S-phase toxin exposure. hus1 is required for wild-type survivorship levels after exposure to the S-phase genotoxin, hydroxyrurea, but not after irradiation. I also made null mutations in the Drosophila brca2 homolog, and demonstrated that brca2 is required for repair by the homologous recombination pathway in mitotically-dividing cells, and for repair of meiotic recombination intermediates. Brca2 was shown to have a second role during meiosis in the efficient activation of the meiotic recombination checkpoint. I observed a physical interaction between Brca2 and Hus1 in ovarian lysate and suggest that Hus1 and Brca2 may have a common role in checkpoint signal transduction. Finally, I found that over-expression of Brca2 in the germline results in sister chromatid separation failure during cycle 1 of embryogenesis and the formation of multi-polar spindles during subsequent cell cycles. Collectively, these results demonstrate that Brca2 is a multifunctional protein that likely has many roles in safeguarding genome stability. |
