DNA replication in protein extracts from human cells requires ORC and Mcm proteins.
We used protein extracts from proliferating human HeLa cells to support plasmid DNA replication in vitro. An extract with soluble nuclear proteins contains the major replicative chain elongation functions, whereas a high salt extract from isolated nuclei contains the proteins for initiation. Among the initiator proteins active in vitro are the origin recognition complex (ORC) and Mcm proteins. Recombinant Orc1 protein stimulates in vitro replication presumably in place of endogenous Orc1 that is known to be present in suboptimal amounts in HeLa cell nuclei. Partially purified endogenous ORC, but not recombinant ORC, is able to rescue immunodepleted nuclear extracts. Plasmid replication in the in vitro replication system is slow and of limited efficiency but robust enough to serve as a basis to investigate the formation of functional pre-replication complexes under biochemically defined conditions.
Baltin
Jens
J
Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Leist
Sandra
S
Odronitz
Florian
F
Wollscheid
Hans-Peter
HP
Baack
Martina
M
Kapitza
Thomas
T
Schaarschmidt
Daniel
D
Knippers
Rolf
R
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D026341:Minichromosome Maintenance 1 Protein / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry; D051738:Origin Recognition Complex; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D004268:DNA-Binding Proteins; D026341:Minichromosome Maintenance 1 Protein; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D011994:Recombinant Proteins