Studies of the Properties of Human Origin Recognition Complex and Its Walker A Motif Mutants

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 1; pp. 69 - 74
• Main Authors: Giordano-Coltart, Jennifer, Ying, Carol Y., Gautier, Jean, Hurwitz, Jerard
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 04-01-2005; National Acad Sciences
• Subjects: Adenosine triphosphatases; Adenosine Triphosphate - metabolism; Amino Acid Motifs - genetics; Amino Acid Motifs - physiology; Animals; Antibodies; Baculoviridae; Binding sites; Biochemistry; Biological Sciences; Chromatin; Cloning, Molecular; Complementary DNA; Deoxyribonucleic acid; DNA; DNA replication; DNA Replication - physiology; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Enzymes; Genetic mutation; Genetic Vectors; Hominids; Humans; Hydrolysis; Mutation; Origin Recognition Complex; Proteins; Viruses; Xenopus; Xenopus laevis; Xenopus Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0408690102

The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. In this report we describe the isolation of the wild-type Homo sapiens (Hs)ORC and variants containing a Walker A motif mutation in the Orc1, Orc4, or Orc5 subunit using the baculo-virus-expression system. Coexpression of all six HsORC subunits yielded a stable complex containing HsOrc subunits 1-5 (HsORC1-5) with virtually no Orc6 protein (Orc6p). We examined the ATPase, DNA-binding, and replication activities of these complexes. Similar to other eukaryotic ORCs, wild-type HsORC1-5 possesses ATPase activity that is stimulated only 2-fold by single-stranded DNA. HsORC1-5 with a mutated Walker A motif in Orc1p contains no ATPase activity, whereas a similar mutation of either the Orc4 or Orc5 subunit did not affect this activity. The DNA-binding activity of HsORC1-5, using lamin B2 DNA as substrate, is stimulated by ATP 3- to 5-fold. Mutations in the Walker A motif of Orc1p, Orc4p, or Orc5p reduced the binding efficiency of HsORC1-5 modestly (2- to 5-fold). Xenopus laevis ORC-depleted extracts supplemented with HsORC1-5 supported prereplication complex formation and X. laevis sperm DNA replication, whereas the complex with a mutation in the Walker A motif of the Orc1, Orc4, or Orc5 subunit did not. These studies indicate that the ATP-binding motifs of Orc1, Orc4, and Orc5 are all essential for the replication activity associated with HsORC.