The origin recognition complex (ORC) plays an essential role in the initiation of DNA replication by binding to origin sequences throughout the cell cycle and acting as a scaffold for the association of additional protein factors in G1 phase (reviewed in Bell, 2002). Originally isolated and characterized in the budding yeast Saccharomyces cerevisiae (Bell and Stillman, 1992), ORC is composed of six distinct subunits, and orthologs of each have now been found in a wide range of eukaryotic species (reviewed in DePamphilis, 2005). In early G1 phase, ORC promotes the origin-association of the clamp loading protein Cdc6 in an ATP-dependent manner (Speck et al, 2005). Another factor, Cdt1 (Devault et al, 2002; Tanaka and Diffley, 2002) directs the nuclear import of the MCM (minichromosome maintenance) family of proteins, Mcm2–7, which act as replication fork helicases (reviewed in Bell and Dutta, 2002). Once ORC and Cdc6 are present at origins, Cdt1–Mcm2–7 can also associate with origin DNA. Hydrolysis of ATP by Cdc6 is then thought to result in Cdt1 dissociation and a stronger Mcm2–7 binding at origins. Subsequent ATP hydrolysis by ORC catalyzes the loading of additional Mcm2–7 complexes (Kawasaki et al, 2006; Randell et al, 2006). Once tightly bound, the continued association of at least Mcm2 with chromatin in G1 phase requires the presence of another ORC-associated protein, Mcm10 (Homesley et al, 2000). Collectively, this assemblage of proteins is known as the pre-replicative complex (pre-RC). In addition to pre-RC formation, the initiation of DNA replication requires the activation of two kinase complexes, Clb5/Cdc28 and Dbf4/Cdc7, which promote the origin-association of Cdc45 (Nougarede et al, 2000; Zou and Stillman, 2000). Cdc45 in turn recruits DNA polymerases to origins (Mimura and Takisawa, 1998; Aparicio et al, 1999; Zou and Stillman, 2000).
Curiously, only five of the six ORC subunits are required for origin recognition and binding in vitro (Lee and Bell, 1997). Even though Orc6 is an essential protein in budding yeast (Li and Herskowitz, 1993), it appears to be dispensable for these functions and its role in cell cycle progression has yet to be determined. Clearly, Orc6 association with the other budding yeast ORC subunits suggests a function in DNA replication. Li and Herskowitz disrupted one copy of ORC6 in a diploid yeast strain and, following sporulation, were able to observe up to two of rounds of cell division from spores inheriting the ORC6 knockout. Arrested cells had a large budded phenotype often observed for DNA replication mutants, but the stage of cell cycle arrest could not be determined by FACS analysis due to an insufficient number of cells. Studies involving the replication of Xenopus sperm DNA in Drosophila egg extracts indicate that Orc6 can promote DNA replication in this in vitro system (Chesnokov et al, 2001). With human cancer cells, depletion of Orc6 by transfection with siRNA duplexes resulted in a significant reduction in the number of positive cells in BrdU incorporation assays, consistent with a replicative function (Prasanth et al, 2002). As well, research with both human and fruit fly cells point to mitotic and/or cytokinetic functions in addition to a role for Orc6 in DNA replication (Prasanth et al, 2002; Chesnokov et al, 2003).
Here, we demonstrate that Orc6 is required for the initiation of DNA replication in budding yeast cells and is dispensable for progression through mitosis and cytokinesis. We further show that Orc6 is required for the maintenance of MCM protein association with chromatin, and that depletion of Orc6 after pre-RC formation inhibits replication origin firing.