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Updated: Aug 10, 2026

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
DNA replication in protein extracts from human cells requires ORC and Mcm proteins
Jens Baltin1, Sandra Leist, Florian Odronitz
1Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Abstract:
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.
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