The origin recognition complex (ORC) is a six protein assembly that binds S. cerevisiae origins of replication and directs DNA replication throughout the genome and transcriptional silencing at the yeast mating-type loci. Cloning of the genes encoding the 120 kDa (ORC1), 62 kDa (ORC3), and 56 kDa (ORC4) subunits of ORC is reported in this paper and the reconstitution of the complete complex after expression of all six subunits in insect cells. Orc1p is related to Cdc6p and Cdc18p, which regulate DNA replication and mitosis, and to Sir3p, a regulator of transcriptional silencing. The N-terminal region of Orc1p is highly related to Sir3p, and studies of Orc1p/Sir3p chimeric proteins indicate that this domain is dedicated to the transcriptional silencing function of ORC (Bell, 1996).

Constituents of the human replication ORC are in the process of being identified. Of the six subunits of ORC identified in Saccharomyces cerevisiae, homologs of four have been molecularly cloned from human cells and are known to associate with each other. A fifth, 80 kDa protein consistently coimmunopreciptiates with human ORC2 from human cell extracts. The immunoprecipitation reaction was scaled up, and the 80 kDa protein was identified by ion trap mass spectrometry. Multiple peptides were identical to the sequence of the protein encoded by the human EST clone. Additional peptides were identified, however, suggesting a 5' truncation of the clone. A complete cDNA was obtained by RACE-PCR and from other EST clones in the database. The sequence of the 80 kDa protein deduced from the full-length cDNA reveals that it is 30% identical (42% similar) to Drosophila melanogaster Latheo. Accordingly, the 80 kDa protein Homo sapiens has been termed Lat (HsLat). HsLat and DmLat also appear related to S. cerevisiae ORC3. When the human protein is compared with all translated ORFs in the S. cerevisiae genome database, ScORC3 emerges as the closest homolog in the yeast genome, with one region of the human protein (residues 216 to 559) 23% identical (43% similar) to ScORC3 (Pinto, 1999 and references).

Immunoprecipitation of human ORC2 with anti-HsORC2 antibodies coprecipitates metabolically labeled HsORC4 (45 kDa) and an unknown protein of 80 kDa (Quintana, 1997). In a complementary experiment, anti-HsLat immunoprecipitates were probed with anti-HsORC2 or anti-HsLat. Both proteins were present in the anti-HsLat immunoprecipitate, confirming that HsORC2 and HsLat associate with each other in cell extracts. The interaction between HsORC2 and HsLat is not mediated by association of the two proteins with DNA. The presence of 200 µg/ml ethidium bromide, which intercalates with DNA and disrupts protein-DNA interactions does not dissociate the HsORC2-HsLat complex (Pinto, 1999).

HsORC4 and HsORC5 were expressed in mammalian cells with an N-terminal glutathione S-transferase (GST) epitope tag. Both GST-HsORC4 and GST-HsORC5 expressed in mammalian cells copurify with human HsORC2 (Quintana, 1997; Quintana, 1998). When GST-HsORC4 or GST-HsORC5 is isolated from human embryonic kidney 293T cells by affinity purification with glutathione agarose beads, HsLat copurifies. GST alone expressed in mammalian cells failed to copurify with HsLat, suggesting that HsLat forms a complex in mammalian cells with HsORC4 and HsORC5, as it does with HsORC2. GST-HsLat also was expressed in mammalian cells and shown to copurify specifically with the 72 kDa HsORC2 protein on glutathione agarose beads. At least a portion of cellular HsLat, HsORC2, HsORC4, and HsORC5, therefore, are associated with each other, which is consistent with the notion that Lat functions as a subunit of the ORC (Pinto, 1999).

The Xenopus origin recognition complex is essential for chromosomal DNA replication in cell-free extracts. The Xenopus origin recognition complex has been purified with anti-Xorc2 antibodies and its composition and properties analyzed. Xorc2 (p63) is specifically associated with Xorc1 (p115) and up to four additional polypeptides (p81, p78, p45, and p40). The cDNA encoding p81 is highly homologous to various expressed sequence tags from humans and mice encoding a protein of previously unknown function. p81 bears sequence resemblence to Drosophila Latheo (Pinto, 1999). Immunodepletion of p81 from Xenopus egg extracts, which also results in the removal of Xorc2, completely abolishes chromosomal DNA replication. Thus, p81 appears to play a crucial role at S phase in higher eukaryotes (Carpenter, 1998).

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