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

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In eukaryotes, the Dbf4-dependent kinase (DDK) triggers DNA synthesis at origins ofreplication 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 thefilamentous 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 thenimQ 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 bystabilizing nimO18p or by elevating nimO18p levels, whereas snoB suppression may occurthrough direct association with nimO18p. This project is devoted to isolating the snoA and snoBgenes, and to defining the molecular mechanisms by which snoAp and snoBp influence nimOpfunction. 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 snoAand snoB genes; and (2) use cell biological and biochemical tools to investigate mechanisms bywhich snoA and snoB suppressors modify nimO function. For example, in vivo DNA labelingwill be used to measure DNA synthesis, and northern blotting will be used to assess nimOmRNA levels. Epitope-tagged nimO and/or cdc7 asp alleles will be used to characterize nimOpcell cycle dynamics, including turnover, phosphorylation, localization, and the influence of snoAand snoB suppressors. A nimOp-Cdc7p kinase assay will employ the tagged alleles andMcm2p 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.