RUI: How do the snoA and snoB Suppressors Modify Function of the Aspergillus NimODbf4 Initiator of DNA Synthesis?

Gettysburg College, Gettysburg PA

Investigators

Abstract

In eukaryotes, the Dbf4-dependent kinase (DDK) triggers DNA synthesis at origins of replication by phosphorylating at least one component of the replicative DNA helicase, Mcm2p. This in turn leads to unwinding of DNA and establishment of the replication fork. In the filamentous fungus Aspergillus nidulans, DDK is composed of a regulatory subunit, nimO Dbf4 , and a kinase catalytic subunit, cdc7 asp . The substrate of nimOp-cdc7p kinase is encoded by the nimQ Mcm2 gene. Mutations in two newly identified genes, snoA and snoB (suppressor-of-nimO), partially alleviate the heat sensitivity of the nimO18 mutation. snoA suppressors act indirectly by stabilizing nimO18p or by elevating nimO18p levels, whereas snoB suppression may occur through direct association with nimO18p. This project is devoted to isolating the snoA and snoB genes, and to defining the molecular mechanisms by which snoAp and snoBp influence nimOp function. The specific aims of the research are as follows: (1) use a combination of walking, sequencing, and functional complementation to isolate and molecularly characterize the snoA and snoB genes; and (2) use cell biological and biochemical tools to investigate mechanisms by which snoA and snoB suppressors modify nimO function. For example, in vivo DNA labeling will be used to measure DNA synthesis, and northern blotting will be used to assess nimO mRNA levels. Epitope-tagged nimO and/or cdc7 asp alleles will be used to characterize nimOp cell cycle dynamics, including turnover, phosphorylation, localization, and the influence of snoA and snoB suppressors. A nimOp-Cdc7p kinase assay will employ the tagged alleles and Mcm2p nimQ as a substrate. This project will enhance the understanding of eukaryotic DNA synthesis and cell cycle regulation by defining the functions of two genes that interact with an initiator of DNA synthesis. These discoveries may in turn provide new insights about cell division. In addition, this project will provide undergraduate students with the opportunity to play an active, substantive role in an ongoing, scientifically rigorous research project.