The Orc4p and Orc5p Subunits of the Xenopus and Human Origin Recognition Complex Are Related to Orc1p and Cdc6p*
- Tamara Tugal‡**,
- X. Helena Zou-Yang§**,
- Kimberley Gavin§,
- Darryl Pappin¶,
- Benito Canas¶,
- Ryuji Kobayashi§,
- Tim Hunt‡ and
- Bruce Stillman§‖
- From the ‡Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom, the ¶Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom, and the §Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
Abstract
The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides (∼110, 68, 64, 48, 43, and 27 kDa) copurified withXenopus Orc1p. Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p. Microsequencing of the 43- and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively. Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits. Human ORC5 was also cloned; its sequence displayed extensive homology to bothDrosophila and yeast ORC5. Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p. Finally, we present the sequence of the putative Xenopus and human Orc3p.
Footnotes
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↵* This research was supported by National Institutes of Health Research Grant CA13016 (to B. S.) and by a grant from the Darwin Trust of Edinburgh (to T. T.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) (human Orc4p), (mouse Orc4p), (Xenopus Orc4p), and (human Orc5p).
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↵‖ To whom correspondence should be addressed: P. O. Box 100, Cold Spring Harbor, NY 11724. Tel.: 516-367-8383; Fax: 516-367-8879.
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↵** The first two authors contributed equally to this work.
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↵2 I. Chesnokov and M. Botchan, personal communication.
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↵3 H. Yang, K. Simpson, M. Hidaka, and B. Stillman, unpublished results.
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↵4 A. F. Neuwald, L. Aravind, J. L. Spouge, and E. V. Koonin, submitted for publication.
- Abbreviations:
- ORC
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origin recognition complex
- MES
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4-morpholineethanesulfonic acid
- PAGE
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polyacrylamide gel electrophoresis
- EST
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expressed sequence tag
- ORF
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open reading frame
- mAb
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monoclonal antibody
- CSF
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cytostatic factor.
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- Received July 28, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
