We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nucleaseresistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.
The human replication protein Cdc6p is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle. However, the amounts of Cdc6p on chromatin remain high during S phase implying either that displaced Cdc6p can rebind to chromatin, or that Cdc6p is synthesized de novo. We have performed metabolic labeling experiments and determined that [ 35 S]methionine is incorporated into Cdc6p at similar rates during the G1 phase and the S phase of the cell cycle. Newly synthesized Cdc6p associates with chromatin. Pulse-chase experiments show that chromatin-bound newly synthesized Cdc6p has a half life of 2-4 h. The results indicate that, once bound to chromatin, pulse-labeled new Cdc6p behaves just as old Cdc6p: it dissociates and eventually disappears from the nucleus. The data suggest a surprisingly dynamic behaviour of Cdc6p in the HeLa cell cycle.Keywords: cell cycle; DNA replication; hCdc6; phosphorylation; turn-over.The eukaryotic replication initiation protein Cdc6 (Cdc6p) is a member of the large AAA + family of ATPases [1]. Like other members of this family, Cdc6p possesses a bipartite purine nucleoside triphosphate binding domain consisting of the conserved Walker A and Walker B motifs. In addition, Cdc6p contains several potential phosphorylation sites in the N-terminal region. Cdc6p is required for the formation of pre-replicative complexes and therefore essential for replication initiation in eukaryotic cells.Pre-replicative complexes are assembled in a stepwise manner during the G1 phase of the eukaryotic cell cycle. Cdc6p associates with the chromatin-bound six-subunit origin recognition complex (ORC) and promotes, together with the Cdt1 protein [2,3], the subsequent loading of the Mcm protein complex. The fully assembled pre-replicative complex is induced to activate replication origins by at least two classes of protein phosphorylating enzymes, cyclindependent kinases (Cdk) and the Dbf4-Cdc7 kinase [4][5][6].In yeasts, Cdc6p is expressed during the G1 phase [7,8], associates with stationary ORC [9,10] and loads Mcm initiation proteins in reactions requiring an intact nucleotide binding domain [11][12][13]. Once replication begins, yeast Cdc6p is phosphorylated and then rapidly destroyed by ubiquitin-mediated protein degradation. The regulated destruction of Cdc6p effectively prevents the binding of Mcm proteins, and therefore prevents the re-replication of DNA sections that had already replicated during the same S phase [14][15][16][17][18][19][20][21][22]. In fact, overexpression of the wild-type Cdc6p homolog (cdc18) in the yeast Schizosaccharomyces pombe [17], and certain mutant alleles of the Saccharomyces cerevisiae gene CDC6 induce the repeated activation of replication origins within one cell cycle [23]. Normally, however, the amounts of Cdc6p fluctuate across the yeast cell cycle. They rapidly decrease with the entry of yeast cells into S phase and increase again during the following G1 phase with the synthesis of new Cdc6p.In contrast, the rapid S...
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