We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches. These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdcl3 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S. pombe cyclin-B homolog cigl, which appears to play a role in G1 or S phase. CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the Gl-specific cyclin, CLN1. Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clbl,2 and clb2,3 cells were inviable. Using clbl,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase. CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle. Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.

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