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Two new members of the mouse origin recognition complex (ORC) have been cloned that are closely related to Saccharomyces cerevisiae ORC4 and ORC5 as well as to their human homolog. Both MmORC4p and MmORC5p have a putative nucleotide triphosphate binding motif. Transcription of MmORC4 and MmORC5 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. The transcription levels of both ORC genes are constantly high in all phases of the cell cycle. A screen based on the two-hybrid approach suggests that the product of the ORC4 gene interacts with the ORC2, but not with the ORC1 protein. The conservation of structure among members of the ORC4- and ORC5-related family of proteins suggests that these proteins play a key role in the initiation of DNA replication in all eukaryotes.

The kinase Dbf4p/Cdc7p is required for the G1/S phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae. Here, we report the identification and characterization of mouse and human cDNAs whose products are related in sequence to Saccharomyces cerevisiae DBF4 cDNA. Both mammalian Dbf4 proteins contain a putative site for phosphorylation by CDK, PEST protease cleavage sites, nuclear localization signals and a short-looped zinc finger-like domain. Transcription of MmDBF4 is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. Upon replenishment of the medium, transcript levels increase during progression through G1, peaking as cells enter S phase. MmDbf4p interacts physically with Cdc7p and Mcm2p in vivo. Using fluorescence in situ hybridization (FISH), the human DBF4 gene was localized to chromosome 7 (q21.3), whereas FISH mapped the murine counterpart to band A2 on chromosome 5. The results of chromosome mapping indicate that in both mouse and human the gene is present as a single copy. The structural conservation between Dbf4-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.