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.The assembly of pre-replication complexes (pre-RC) 6 on mammalian genomes has been well described. As in other eukaryotes, it begins with the binding of an origin recognition complex (ORC) to chromatin. ORC consists of a stable core complex including subunits Orc2-Orc5 associated with less stably bound subunits Orc1 and Orc6 (1-4). Chromatinbound ORC recruits two other proteins, Cdc6 and Cdt1, of which Cdc6 stabilizes the binding of ORC (5) and allows Cdt1 to load the hexameric Mcm complex (composed of Mcm2-Mcm7) (reviews in Refs. 6 -9).At the beginning of S-phase, pre-RCs are converted into active replication forks under the guidance of protein kinases Cdc7 and Cdk2. This process requires the origin binding of several additional initiation factors, including Mcm10 and Cdc45, which together initiate a series of events that ultimately lead to origin unwinding, recruitment of replicative DNA polymerases, and the establishment of replication forks (6).Although the order of the reactions appears to be well established, many mechanistic details have yet to be worked out, and for that purpose it would be useful to possess an in vitro replication system with completely soluble proteins and a biochemically amenable DNA template. A highly successful replication system with soluble constituents is based on extracts from Xenopus eggs. The key components are a membrane-free cytoplasmic extract (for the chain-elongating functions) and a highly concentrated nuclear extract (for initiator proteins) (10). The system replicates either frog sperm chromatin or protein-free plasmid DNA and has been and still is very successful in investigations into the events leading to pre-RC formation (11-15) and to characterize important replication factors such as Cdt1 and Mcm8 (16,17). The Xenopus egg extract contains high amounts of initiator proteins such as ORC and Mcm proteins, stored for subsequent rounds of cell divisions in early embryogenesis. It therefore differs from the situation in proliferating adult mammalian cells with their limited amounts of ORC.In vitro replication systems with cytosolic mammalian cell extracts, using simian virus 4...