FIG. 4.

Replication of PyV DNA by chimeric pol-prim complexes. (A) Increasing amounts (0.25 and 0.5 U of DNA polymerase) of recombinant pol-prim containing murine p180, p68, p58, and chimeric p48 (lanes 3 to 18), four mouse subunits (lanes 19 to 22), or four human subunits (lanes 23 to 26) were added to depleted human 293S cell extracts (lanes 1 and 2) supplemented with PyV Tag and a PyV origin-containing plasmid. (B) The replication products of these extracts were also determined with recombinant enzymes containing three human subunits and hybrid p48. The assays were carried out in the absence of recombinant pol-prim (lanes 1 and 2) or in the presence of recombinant mouse (lanes 3 to 6), human (lanes 23 and 24), and hybrid pol-prim containing the three human subunits p180, p68, and p58, together with a chimeric subunit p48 (lanes 7 to 22). DNA synthesis products for panels A and B were analyzed for complete DNA replication by digestion with 10 U each of EcoRI and DpnI (even-numbered lanes). In parallel, the products were linearized with 10 U of EcoRI (odd-numbered lanes). The DNA synthesis products were visualized by autoradiography. The arrow at the right side of the figure indicates the linearized DNA synthesis products. (C) The incorporation of dNMPs into PyV DNA was determined by acid precipitation of DNA and scintillation counting (light and dark columns, showing means and standard deviations of a minimum of three replication assays with 0.25 and 0.5 primase units, respectively, of the indicated pol-prim). Columns 1, negative control of depleted extracts; columns 2, mouse pol-prim; columns 3 to 10, hybrid pol-prim as indicated; columns 11, human pol-prim.