Studies of the properties of human origin recognition complex and its Walker A motif mutants.
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 baculovirus-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.
Giordano-Coltart
Jennifer
J
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 97, New York, NY 10021, USA.
Ying
Carol Y
CY
Gautier
Jean
J
Hurwitz
Jerard
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000818:Animals; D016367:Baculoviridae; D003001:Cloning, Molecular; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D051738:Origin Recognition Complex; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; C497223:ORC1 protein, human; C497226:ORC5 protein, human; C497227:ORC6 protein, human; C497222:Orc4 protein, Xenopus; D051738:Origin Recognition Complex; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate