In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G 1 and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.The recent identification of multiple eukaryotic proteins that bind directly or indirectly to origins of DNA replication has set the stage for a thorough exploration of how DNA replication is initiated and regulated in eukaryotes. Central to the process is the origin recognition complex (ORC), a tight complex of six polypeptides identified initially in Saccharomyces cerevisiae because it binds in a sequence-specific manner to origins of DNA replication and is involved in the initiation of chromosomal DNA replication (1,3,27). Homologs of three of the subunits of the ORC (Orc1, Orc2, and Orc4) have been identified in humans (16,32) and in other eukaryotes (14,17,22,28).An additional molecule, CDC6 in S. cerevisiae and Cdc18 in Schizosaccharomyces pombe, is essential for the onset of DNA replication and known to associate physically with the ORC and cdc2 kinase (2,12,15,18,22,24,37). The yeast CDC6/ Cdc18 proteins decrease in concentration as cells proceed through S phase, with overexpression of Cdc18 in S. pombe resulting in the rereplication of DNA in G 2 (29). The Xenopus CDC6/Cdc18 and ORC are required for DNA replication and for the loading of the minichromosome maintenance (MCM) proteins onto the chromatin (6,11,13,19,34,35). Collectively, a model has emerged that emphasizes the central role of CDC6/Cdc18 in cooperating with ORC and MCM proteins to form a prereplication complex at origins of DNA replication in G 1 . Once replication begins, the concordant removal of CDC6/ Cdc18 prevents the loading of MCM proteins onto the originbound ORC in G 2 , thereby preventing rereplication of DNA. After mitosis, synthesis of new CDC6/Cdc18 protein allows the loading of MCM proteins on the chromatin, perhaps by forming a bridge between the chromatin-bound ORC and the MCM proteins (4). The prereplication initiation complex thus formed in G 1 is ready to initiate DNA replication upon the activation of S-phase-promoting factors like cyclin-cdk's and CDC7 kinase.We identified and cloned...

A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situ hybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast.