Role of cdc21+ and related genes in eukaryotic chromosome replication

The Schizosaccharomyces pombe cdc21+ gene product is related to the Mcm2-3-5 family of replication proteins. By phylogeny analysis of their protein sequences and screening for cdc21+-related sequences using molecular probes I have suggested that at least six types of cdc21+-related genes may be present in the yeast genome. The isolation of interaction suppressors of the cdc21ts mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11+ and dom1+, whose overexpression specifically affected the viability of cdc21ts cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein. I have studied the phenotype of S. pombe cells overexpressing the cdc21+ gene and amino-terminal truncations of it. Overexpression of the cdc21+ gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21+ arrested in S phase and also entered mitosis in the absence of nuclear division. The possibility that chromosomes in cdc21ts arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21ts cells arrest in S phase and may contain DNA damage. I have generated two new mutant alleles of cdc21+. The first allele was made by deleting most of the cdc21+ open reading frame (ORF). The second allele was constructed by placing the cdc21+ ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.