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.
Highlights
At the beginning of S-phase, pre-RCs are converted into active replication forks under the guidance of protein kinases Cdc7 and Cdk2
In vitro replication systems with cytosolic mammalian cell extracts, using simian virus 40 (SV40) DNA as template, and a viral protein, T antigen, as initiator have been used since the mid-1980s (18 –21) and have been extremely successful in identifying many of the mammalian proteins required for replicative chain elongation
Plasmid DNA is efficiently replicated in vitro when it contains the SV40 origin of replication and when the viral protein T antigen is used as an initiator
Summary
At the beginning of S-phase, pre-RCs are converted into active replication forks under the guidance of protein kinases Cdc and Cdk. Optimal DNA synthesis in these systems requires a cytosolic extract and a high salt nuclear extract and allows semiconservative replication of plasmid DNA (with eukaryotic DNA inserts), to an extent that is only 1–3% of that achieved with T antigen and SV40 DNA under similar biochemical conditions This is quite plausible because the initiator T antigen is added in high amounts to the incubation mixtures and does not need to interact with other proteins for the recognition and the unwinding of the viral origin. The dozen or so cellular initiator functions are inevitably diluted when extracted from the nuclei of cultured cells and assemble on one of many possible sites on the plasmid template where they must interact in a highly complex manner before they are ready to initiate a replication round With these limitations in mind we decided to investigate whether ORC and other components of the pre-RC are necessary for replication initiation in vitro. The main conclusion was that the in vitro system appeared to be sufficiently robust to investigate questions related to replication initiation in mammalian cells
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