The ORC1 Cycle in Human Cells

Abstract

Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G 1 phase, reaches a peak at the G 1 /S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring.The replication of chromosomal DNA in eukaryotes is limited to once per cell division cycle. This control appears to be achieved mainly by the regulation of replication origins so that they fire only once per cell cycle. The origin recognition complex (ORC), 1 identified in budding yeast as a protein complex that binds origins, consists of six gene products (1-5). In addition to ORC, several factors highly conserved among eukaryotes are involved in initiation (6 -8). The sequential assembly of these factors on origin-ORC complexes precedes initiation, as has been shown in yeast and Xenopus systems. For example, minichromosome maintenance (MCM) proteins are loaded onto origins in the presence of ORC and CDC6, which establishes the pre-replicative complex (pre-RC) necessary for subsequent protein assembly (9 -13). After origin firing, the pre-RC changes to a post-replicative form by the dissociation of MCM from the complex (12, 14, 15). These associations provide an important mechanism that 1) ensures that replication origins fire at precise times and 2) prevents re-initiation. Budding yeast ORC is a static complex that is maintained at a constant level and remains bound to origins throughout the cell cycle (4, 5). Thus, MCM loading in yeast is mainly regulated by other factors such as cell cycle-regulated Cdc6 or CDK kinase activities (7,8,16). It is also known that the phosphorylation status of ORC subunits correlates with the timing of pre-RC formation, suggesting a role for ORC phosphorylation in MCM loading (16). Similar phosphorylation of ORC subunits was found in a Xenopus egg extract system, suggesting a conserved mechanism for the regulation of ORC functions (17, 18). Recent studies have elucidated possible...

The ORC1 Cycle in Human Cells

“…1B). In this experiment, the level of ORC1 was low in metaphase and early G 1 (0 -3 h), rose between 6 and 12 h (middle G 1 ), reached a peak at 12 h (G 1 /S), and then decreased from S to G 2 phase, slightly in advance of the decrease in the level of cyclin E, as described in a previous study (31) (Fig. 4A).…”

“…Cell Culture and Synchronization-Cell culture, synchronization, and monitoring of the cell cycle were done as described (31,32). Construction of the 293 cell line expressing FLAG-ORC1 (1C2) was also described (31).…”

“…Immunoprecipitation and Immunoblotting-All antibodies used in this study have been described (31). #209 (anti-ORC1 antibody) beads were prepared by immobilization of the purified antibody to protein A-Sepharose (Amersham Biosciences, UK), at a ratio of 2 g of IgG to 10 l of beads, with dimethyl pimelimidate (Sigma, St. Louis, MO).…”

“…Fractionation of Cellular Proteins-Whole cell extract preparation and fractionation of proteins from HeLa S3 cells with Triton X-100 (Triton), NaCl, or DNase I were done as described (31,32).…”

Cell Cycle Dependence of Group VIA Calcium-independent Phospholipase A2 Activity

“…Cell cycle-dependent mechanisms of proteolysis are reported to be largely dependent on the 26 S proteosome which proteolyzes polyubiquitinated proteins (40). Of particular interest is the recent observation that catabolism of the largest subunit of the human origin recognition complex (ORC1) during S phase is mediated through the SCF skp2 ubiquitin ligase complex (46). Hence it is possible that like ORC1, the truncated iPLA 2 proteins are phosphorylated by S phase checkpoint kinases (e.g.…”