ATP-dependent assembly of the human origin recognition complex.
The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.
Siddiqui
Khalid
K
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Stillman
Bruce
B
D000255:Adenosine Triphosphate / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D004261:DNA Replication; D006801:Humans; D008954:Models, Biological; D051738:Origin Recognition Complex / Q000378:metabolism*; D018741:Replication Origin; D016196:S Phase
D018797:Cell Cycle Proteins; C497223:ORC1 protein, human; C497224:ORC2 protein, human; C497225:ORC3 protein, human; C109152:ORC4 protein, human; C497226:ORC5 protein, human; C497227:ORC6 protein, human; D051738:Origin Recognition Complex; D000255:Adenosine Triphosphate