The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p

The location of origins of DNA replication within the Saccharomyces cerevis iae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebr ate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodie s were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides (simi lar to 110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p, Prot ein sequencing also showed the 64-kDa protein to be the previously identifi ed Xenopus Orc2p. Microsequencing of the 43- and 48-kDa proteins that copur ified with Orc1p and Orc2p led to their identification as the Orc4p and Orc 5p subunits, respectively. Peptide sequences from the 43-kDa protein also a llowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 s ubunits, Human ORC5 was also cloned; its sequence displayed extensive homol ogy to both Drosophila and yeast ORC5. Surprisingly, comparison of the amin o acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structur ally related to each other and to the replication initiation protein, Cdc6p . Finally, we present the sequence of the putative Xenopus and human Orc3p.