Genetic Analysis of Human Orc2 Reveals Specific Domains That Are Required in Vivo for Assembly and Nuclear Localization of the Origin Recognition Complex

Abstract

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

“…Affinity Purification of Proteins-Purification of FH-Orc6 proteins was carried out by a two-step affinity purification protocol, as described previously (9). In brief, FH-Orc6 protein was purified from TK100 extracts of HeLa-RR cells.…”

“…Cells-The human cell lines HeLa (#CCL-2, American Type Culture Collection) and HeLa-RR and their culture conditions have been described (9). These cells were synchronized as they entered S-phase by culturing them in the presence of 4 mM deoxy-Thd for 18 h, then in fresh medium for 12 h, and again with deoxy-Thd for 16 h. Cells were arrested in metaphase by culturing them in the presence of deoxy-Thd for 18 h and then releasing them into 100 ng/ml nocodazole for 8 h. HeLa-RR lines that constitutively expressed either wild-type or mutant Orc6 genes in the absence of continual selection of a genetic marker were constructed as previously described (9).…”

“…When FHOrc6 was used as the affinity tag, ORC(1-6) was purified from the GN50 extract by the two-step affinity purification protocol (9), except that GN buffers were used. When FH-Orc1 was used as the affinity tag, the GN50-extracted HeLa-RR cell pellet was extracted further with GN300 (300 mM NaCl instead of 50 mM NaCl) to maximize recovery of Orc1 protein.…”

“…Immunofluorescence-Cytology was done as previously described (9), except that mouse anti-FLAG antibody (Sigma) was diluted 1:1500 in PBS, and rabbit anti-Orc6 antibody was diluted at 1:1200 in PBS. Images were merged using either MetaVue software (Universal Imaging) or Adobe Photoshop.…”

“…ORC isolated from frog eggs lacks the Orc6 subunit, which is not essential for DNA replication (6). ORC isolated from human cells also lacks the Orc6 subunit (7)(8)(9)(10), and only the human ORC (1)(2)(3)(4)(5) complex is required to initiate DNA replication in an ORC-depleted frog egg extract (9,10). Human ORC (2)(3)(4)(5) binds to DNA and initiates DNA replication only when associated with Orc1 (9,10,26).…”