Identification and characterization of a mouse homolog to yeast Cdc6p

Periodic expression of the Cdc6 protein is essential for the entry of buddi ng yeast cells into S phase, and also for participating in checkpoint contr ols that ensure that DNA replication is completed before mitosis is initiat ed. We have identified a mouse protein closely related to Cdc6p (MmCdc6p) a s well as to its human and Xenopus homologs. The gene coding for MmCdc6p (C dc6) is located at band D on murine chromosome 11. Analysis of its genomic region revealed that the 13-kb Cdc6 gene is divided into 12 exons by 11 int rons. MmCdc6p has putative cyclin-dependent phosphorylation sites, a destru ction box, nuclear localization signals, a nucleotide triphosphate-binding motif, and a potential leucine zipper. None of these consensus motifs excep t the leucine-zipper and the destruction box overlaps an intron. Expression of MmCdc6 mRNA and protein is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. Upon replenishment of the medium, transcript and protein levels increase during progression through G(1), peaking as ce lls enter S phase. MmCdc6p is phosphorylated in vitro by cdk1/cyclin B, cdk 4/ cyclin D, cdk2/cyclin E, and cdk2/cyclin A, respectively at serine-resid ues. In vivo however, phosphorylation of MmCdc6p is carried out by cdk2/cyc lin A at serine-residues exclusively. Conservation of structures among memb ers of the Cdc6-related proteins suggests that these proteins play a key ro le in the regulation of DNA replication during the cell cycle in all eukary otes. These results strongly suggest, that Cdc6p plays an important role in cell cycle regulation and replication licensing.