Subdomain analysis of the BRDF checkpoint motif of the nimODbf4 regulator of DNA synthesis.

Trevor KerstetterKerstetter
Advisor - Steven James

In the filamentous fungus Aspergillus nidulans, nimODbf4 constitutes the regulatory subunit of the conserved Dbf4-dependent kinase (DDK). In all Dbf4 orthologs the N-terminus harbors a checkpoint domain, the BRCT and DbF4-similarity domain (BRDF). nimODbf4 mutants lacking the BRDF motif (nimO¿BRDF) become sensitive to a wide range of DNA damage agents, and surprisingly, arrest meiosis early in prophase I. We identified a gene, snoA (suppressor-of-nimO), loss of which rescues both of these nimO¿BRDF defects. snoA is orthologous with Rif1, a conserved eukaryotic gene that, in mammals, plays a role in a S-phase DNA damage checkpoint and in the repair of double strand breaks (DSBs). Not known for any Dbf4 ortholog is whether the BRDF checkpoint and meiotic functions are separable into different subdomains. Based upon the predicted secondary structure of this domain, we are using a rational approach to eliminate individual BRDF subdomains. We are creating five different in-frame subdomain deletions and will test these nimODbf4 alleles in snoA+ and snoA mutant backgrounds for their effects on DNA damage responses and meiotic proficiency.