Previous studies have shown that changes in the affinity of the hamster Orc1 protein for chromatin during the M-to-G 1 transition correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites. Here we show that Orc1 is selectively released from chromatin as cells enter S phase, converted into a mono-or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G 1 transition. Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination. In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination. Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase. Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs. The mechanism for initiation of DNA replication is highly conserved among eukaryotes in that homologues of the pro-teins involved in assembly and activation of prereplication complexes (pre-RCs) in the budding yeast Saccharomyces cer-evisiae are found in all other eukaryotes examined so far, and the sequence of events by which these proteins initiate DNA replication is remarkably similar throughout the eukaryotic kingdom (reviewed in references 4 and 20). Nevertheless, the first step in regulating this process appears to differ signifi-cantly between yeasts and mammals. The first step in the initiation of eukaryotic DNA replication is the assembly of a six-subunit origin recognition complex (ORC) at specific sites distributed throughout the genome. For yeast, these sites coincide with genetically defined replication origins (replicons). Analogous sites have been identified for mammals, although they appear to be more complex (dis-cussed in references 2, 10, 31, and 41). All six ORC subunits are required for the initiation of DNA replication of yeast, and Orc1 and Orc2 have been shown to be required for Drosophila and Xenopus (6, 24, 44, 45). In the yeasts S. cerevisiae (11, 17, 27) and Schizosaccharomyces pombe (22, 29), both DNA foot-printing and immunoprecipitation analyses reveal that a com-plete ORC binds to the replication origins immediately after the initiation of replication occurs and it remains stably bound throughout the cell division cycle. Thus, the first step in regu-lating the assembly of pre-RCs in yeast is believed to occur by regulating the activity of Cdc6p/Cdc18p (20) and Cdt1p (51), two proteins that are required for loading Mcm proteins onto ORC-chromatin sites. However, the situation appears quite different in the Metazoa. In multicellular eukaryotes such as frogs, flies, and mam-mals, the affinity of one or more ORC subunits for chromatin changes at specific times during the cell cycle. In Xenopus, Orc proteins in activated eggs bind to sperm chromatin, whereas Orc proteins in meiotic eggs do not (7, 16, 19, 46), and both Orc1 and Orc2 proteins are present on chromatin in cultured Xenopus cells during interphase but not during metaphase (44). In Drosophila, Orc2 remains bound to chromosomes throughout the cell cycle (36), whereas the amount of nuclear bound DmOrc1 is greatest during late G 1 and S phases (3), suggesting a cell cycle-dependent, differential association of DmOrc proteins with chromatin. In mammals, the total amounts of Orc1 and Orc2 present throughout the cell cycle are constant (33, 34, 43, 47), but Orc1 is selectively released from chromatin during M phase (34) and S phase (23). This accounts for the fact that M-phase chromatin does not contain functional ORCs (34, 59) and for the disappearance of an ORC-like footprint at the human lamin B 2 origin during mi-tosis (1). ORC activity is restored during the transition from M to G 1 phase, concomitant with the appearance of Orc1 tightly bound to chromatin and the assembly of pre-RCs at specific genomic sites (25, 34). Therefore, it appears that multicellular eukaryotes regulate the initiation of DNA replication through cell cycle-dependent changes in ORC activity, the first step in the assembly of pre-RCs. In mammals, this appears to be accomplished through selective release of Orc1 from chroma-tin bound ORCs during the S-to-M phase transition, followed by reassembly of functional ORC-chromatin sites during the M-to-G 1 phase transition. This would allow mammals to delay assembly of pre-RCs until both DNA replication and mitosis are completed and a nuclear membrane is assembled. Here we provide further evidence in support of this