Fig. 8. In vitro replication activity of the CHO K1 and xrs-5 cell extracts. (A) Typical autoradiograph of DNA replication products. p186 was incubated in reaction mixtures containing CHO K1 or xrs-5 total cell extracts. The DNA was purified, concentrated and a sample was digested with 1 unit of DpnI for 1 hour at 37°C. The DpnI-digested (lanes 1-12) samples were subjected to electrophoresis on 1% agarose gel. The supercoiled (I), relaxed circular (II) and linear (III) forms of the plasmid and the DpnI-digestion products are indicated. Duplicate samples are shown. (B) xrs-5 nuclear cell extracts do not replicate p186. In vitro DNA replication assays were performed with CHO K1 or xrs-5 nuclear and cytoplasmic (NC), cytoplasmic (C) or nuclear (N) cell extracts. Quantification of DNA replication activities of the cell extracts was done relative to the CHO K1 NC reaction. Each bar represents the average of four experiments and one standard deviation is indicated. (C) Ku restores replication activity to the xrs-5 nuclear cell extracts. In vitro DNA replication assays were performed with either K1 or xrs-5 nuclear cell extracts in the presence of A3/4-affinity-purified Ku. An autoradiograph of the replication products DNA forms II and III are indicated. Lane 1 and 4, 0 ng affinity-purified Ku (OBA); lane 2 and 5, 160 ng affinity-purified (OBA); lane 3 and 6, 600 ng affinity-purified (OBA). (D) Quantification of DNA replication activities of the cell nuclear extracts, relative to the CHO K1 N reaction using 0 ng of OBA, as described in C. Each bar represents the average of three experiments, and one standard deviation is indicated.