CHARACTERIZATION OF A NOVEL CDC GENE (ORC1) PARTLY HOMOLOGOUS TO CDC6OF SACCHAROMYCES-CEREVISIAE

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identic al to that of purified ORC1 protein. Disruption of the gene and subseq uent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cy cle progression of the mutant cells by FAGS after the removal of galac tose from the medium, and microscope observations of cells and their n uclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked com pletely in the cells that had progressed to the S phase under conditio ns deficient in ORC1 protein followed by cell death. Two-dimensional g el analysis of the replication intermediates after the galactose remov al revealed that the depletion of ORC1 protein caused a decrease in th e frequency of initiation of chromosomal replication, eventually resul ting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discuss ed in light of current information on ORC.