Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.
The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.
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Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009154:Mutation; D051738:Origin Recognition Complex; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497215:ORC2 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins