Origin Recognition Complex (Molecular Biology)

DNA replication in bacteria or viruses starts at a specific region of their chromosome, the replication origin, and is triggered by formation of a DNA-protein complex, called the replicator, at that distinct site. In general, the DNA-binding protein called initiator recognizes the specific DNA sequence at the replication origin and nucleates formation of the DNA-protein complex in an ATP-dependent manner. Although it has been difficult to identify such a specific DNA-protein interaction for DNA replication in most eukaryotes, studies with a simple eukaryote, Saccharomyces cerevisiae (yeast), revealed that eukaryotes will also have the same protein-DNA interaction during initiation of DNA replication. The replication origins in yeast chromosomes have been identified as autonomously replicating sequences (ARS), which are defined within short regions by the essential ARS consensus sequence (ACS, the A element) and its accessory elements (the B elements). Many efforts to identify the potential yeast initiator protein as the ARS binding protein have been made, and a multisubunit protein complex called the origin recognition complex (ORC) was finally purified from yeast nuclear extracts (1).

The purified ORC binds to the typical yeast replication origin, ARS1, in the presence of ATP, covering the 11-bp A element consensus sequence and neighboring B1 element. In addition to this ARS-specific binding property, ORC satisfies several other criteria as the yeast initiator protein ((2, 3): (1) ORC binds to all functional ARS. (2) The relative binding of ORC to several mutant ACS parallels their replication activities. (3) Temperature-sensitive mutant forms of ORC subunits result in the temporary arrest of the cell cycle in G1 phase at a nonpermissive temperature and make ARS plasmids unstable even at the permissive temperature. This instability is suppressed by increasing numbers of ARS in the plasmid, suggesting that replication initiation functions are impaired in these mutants. (4) Direct measurement of origin initiation at ARS1 by two-dimensional gel electrophoresis indicated that only a small fraction of ARS1 are active in the temperature-sensitive ORC yeast cells at the permissive temperature; on the other hand, most of the ARS1 functioned in wild-type cells. (5) The ATP-dependence of the ARS1 binding activity is consistent with the requirement of many initiator proteins for ATP in their active form at the origin sequences.

ORC is composed of six different polypeptides, called ORC1 to ORC6, with respective molecular weights of 120, 72, 62, 56, 53, and 50 kDa. All the genes encoding these subunits are essential for the viability of yeast, meaning that none of them are dispensable. In addition to DNA replication, ORC 2 and 5 were identified as factors necessary for the silencing of mating-type gene expression (2, 4), indicating that ORC has dual functions in DNA replication and gene silencing. The primary structures of the ORC polypeptide chains have no similarity to each other, and no typical motifs have been identified, except for strong similarity of ORC1 with one yeast replication gene product, CDC6 (Cdc18 in Saccharomycespombe) (5), and purine nucleotide-binding motifs in ORC1 and ORC5. Klemm et al. (6) actually showed ATP-binding and ATPase activities in ORC regulated by ARS DNA. Binding of ORC to ARS DNA requires the presence of ATP, but not its hydrolysis, and the ARS DNA inhibits the ORC ATPase activity, indicating that ATP will stabilize ORC on the origin DNA. Since the ATP-dependent ORC binding occurs at the prereplicative stage, a mechanism for inducing the ATP hydrolysis of ARS-bound ORC is suggested to activate the replication origin.

In parallel with studies of ORC function, a dynamic protein assembly at the replication origin of yeast was studied by in vivo DNase I footprinting (7). The analysis demonstrated the presence of a specific DNA-protein complex at the ARS1 in vivo. Since the protection pattern against the nuclease digestion in vivo was nearly consistent with that obtained with purified ORC, and functional ORC is required to form the complex, it is most likely that the ORC-ACS complex observed with purified components actually exists at the replication origins in yeast cells. One important observation is a periodic change in the protection pattern in which a protection wider than the simple ORC-ACS complex appears from the end of mitosis to G1 phase and is shifted back to the narrow protection pattern at the beginning of S phase. Since the ORC-ACS protection pattern always exists throughout the cell cycle, it was thought that ORC functions as a landing pad for additional factors, and the periodic change in the protection pattern represents the association of these factors with ORC. Since the wider protection appeared prior to initiation of replication at the replication origin and disappeared upon initiation, it represents formation of the so-called pre-replicative complex, which is defined as a protein assembly to make the replication origin competent for initiation. Several replication initiation-related gene products, CDC6, CDC7, and MCM, a potential licensing factor, are required to form the wider complex at the origin and are thought to be components of the pre-replication complex (8).

Accumulated knowledge about eukaryotic replication proteins inspired the idea that all eukaryotes have the same organization as replication proteins, with conserved primary structures. After the discovery of ORC in yeast, many efforts were made to identify ORC homologues in different species, aiming to find their initiator proteins. Thus far, homologues have been identified in several organisms: S. pombe, Arabidopsis, Drosophila, Xenopus, and humans (9). The ORC2 homologue in Xenopus is necessary to initiate the DNA replication reaction in Xenopus egg extracts (10), and the homologue in Drosophila was genetically identified as a gene required for amplification of the chorion gene locus (11). In addition, ORC subunit homologues in these higher eukaryotes seem to form multisubunit complexes similar to the yeast ORC. Therefore, all eukaryotes may have a conserved mechanism to initiate DNA replication in chromosomes, in which interactions between the counterparts of ORC and ACS will be a central event.

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