Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G1 transition in mammals

Abstract

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G1 phase, and reappeared as cells progressed through G1 phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G1 phase, but became stably bound during mid‐G1 phase, concomitant with the appearance of a functional pre‐replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre‐replication complexes until mitosis is complete and a nuclear structure has formed.

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

“…In order to determine if there were changes occurring at or near the time of the origin decision point (ODP), a G1-phase hallmark at which origins of replication are specified [3,39], we first examined ORC-chromatin association in mitosis and G1-phase using CHO cells synchronized with a brief nocodazole block and release. Consistent with our studies and those of others utilizing the same cell line [3,36,40], we did not detect any significant change in the amounts of ORC1, 2 or 4 in the chromatin-containing fraction during mitosis and throughout G1-phase, although a small amount of ORC1 was consistently found in the soluble fraction during mitosis (Fig. 1A).…”

“…However, this model does not account for all reported results. Several laboratories do not observe fluctuation of total ORC1 levels during the mammalian cell-cycle [3,26,36,37] and in one study, protein synthesis was inhibited throughout the prophase to telophase period, with no effect on the assembly of functional pre-RCs [3]. Importantly, it has been shown that ORCdepleted Xenopus egg extracts can initiate replication within G2-phase Hela cell nuclei [38], which presumably must require the presence of mammalian ORC1 during G2-phase.…”

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…A previous study in CHO cells also observed mitotic chromosome association of CgOrc1 as well as CgOrc4 (39); however, it must be noted that Orc1 shows different cell cycle regulation in these cells compared with human cells. In CHO cells, CgOrc1 is not degraded and stays associated with chromosomes throughout the cell cycle (39); binding of CgOrc1 is weaker during mitosis and stronger in G 1 (89), and inhibition of CDK activity is necessary for CgOrc1 binding to mitotic chromosomes (90). We suggest that the difference between the CHO results and our results with human cells reflects real species-specific differences and not cell line to cell line variation because we have observed similar Orc1 dynamics in multiple human cells lines, including diploid cells.…”

DNA Replication in Protein Extracts from Human Cells Requires ORC and Mcm Proteins

“…an assay with untreated nuclear extract. The rationale for this experiment is that endogenous Orc1 is known to be rapidly degraded in vitro (29,36) with the possible consequence that the amounts of Orc1 in the S300 nuclear extracts were less than optimal for in vitro replication. Indeed, recombinant Orc1 added to the nuclear extract stimulated severalfold the standard replication reaction.…”