Fig. 5. (A) Cell-free DNA replication system. Nuclei (N) prepared from G1 phase WI38 fibroblasts, synchronised by release from quiescence (G0), initiate a single round of semi-conservative DNA replication in cytosolic extracts (C) from S phase HeLa cells substituted with ribonucleoside and deoxyribonucleoside triphosphates (NTPs, dNTPs) and an energy regeneration system (creatine phosphate (CP) and phosphocreatine kinase (CK)). Nuclei are stained with propidium iodide to reveal DNA (red) and with fluorescein-streptavidin (green) to detect biotin-16-dUTP incorporation resulting from in vitro DNA synthesis (Stoeber et al., 1998). Results are expressed as the percentage of nuclei replicating and summarised in the histograms (mean+s.d.). Substitution of nuclear templates and/or extracts with subcellular components from quiescent (G0), terminally differentiated (not shown) or replicative senescent (SEN) cells provides a functional assay for analysis of the mechanisms that establish and/or maintain loss of replicative capacity in ‘out-of-cycle’ cells (B-D). (B) In vitro analysis of the replicative capacity of WI38 G1, quiescent and replicative senescent nuclear templates in either physiological buffer supporting elongation or S phase cytosol. Initiation of DNA replication in vitro is restricted to G1 phase nuclei. (C) In vitro analysis of the replicative capacity of WI38 G1 nuclear templates in quiescent, replicative senescent and S phase cytosolic extracts. S phase cytosolic extracts, but not quiescent or replicative senescent extracts, support efficient in vitro replication. (D) In vitro analysis of the replicative capacity of WI38 G1 nuclear templates in elongation buffer (EB), S phase cytosolic extracts and after titration of senescent cytosolic or nuclear extract into S phase cytosol. Titration of senescent nuclear extract but not nuclear extraction buffer (Buffer) alone resulted in striking inhibition of DNA replication initiation in vitro. Note that different proportions of S phase contaminants in WI38 G1 nuclear preparations in (B, 15%) and (D, <1%) relate to the use of two different batches of G1 nuclei.