Mutations in snoA and snoB(suppressor-of-nimO) act through different mechanisms to rescue defects in the initiation of DNA synthesis.
nimODbf4 of Aspergillus nidulans encodes the regulatory subunit of a conserved eukaryotic enzyme known as Dbf4-dependent kinase (DDK). In budding yeast, Dbf4p initiates DNA synthesis by activating a catalytic subunit, Cdc7p, and escorting it to origins of replication. DDK then triggers origin unwinding through phosphorylation of DNA helicase subunits. Several approaches are underway to investigate nimO andcdc7 function in Aspergillus. For example, cdc7asp was isolated and, by two-hybrid analysis, cdc7pasp was shown to physically associate with nimOp. Additionally, we generated partial nimO18 suppressors in two loci, snoA and snoB (suppressor-of-nimO). snoA/Balleles partially alleviate the temperature sensitivity of nimO18 by restoring growth at 37oC but not 43oC. snoA31 and snoB59 do not interact genetically with ts-lethal mutations in any of 20 cell cycle control genes in the nim (never-in-mitosis) and bim (blocked-in-mitosis) collection. Thus, the suppressors appear to modify only nimO activity or function. The suppressors were tested in an ethanol-dependent, glucose-inviable strain carrying alcA::nimO as the sole nimO gene copy. Recessive snoA alleles rescued growth on glucose, suggesting that snoA mutations may bypass the requirement fornimO. However, by using a parasexual assay we found that instead of bypass, snoA alleles act by stabilizing or enhancing nimOp produced during leakyalcA::nimO expression on glucose. Conversely, semi-dominant snoB alleles could not rescue growth on glucose, suggesting that snoBp and nimOp may associate directly. This hypothesis is supported by the discovery that snoB59 harbors a missense mutation in the cdc7asp gene. Sequencing of cdc7asp from additional snoB alleles is underway to determine if snoB =cdc7asp. (Supported by NSF-RUI# 01-14446 and by a Research and Professional Development Grant from Gettysburg College).
Fungal Genet. Newsl. 50 (Supl):abstract
Full conference title:
22nd Fungal Genetics Conference