FIG. 3.

The wild-type β clamp stimulates Pol I replication in vitro. (A) Reaction mixtures containing M13 ssDNA bearing a 5′-³²P-labeled primer as a template, together with either 0.05, 0.5, or 5 nM of the indicated polymerase, as described in Materials and Methods. After being quenched, aliquots of each reaction mixture were electrophoresed through an 8% urea-polyacrylamide gel. The lanes labeled P represent control reactions lacking clamp and polymerase and serve to indicate the position of free primer. Replication products were visualized by phosphorimager analysis. The faint full-length bands visible in reactions containing β159 and 0.05 or 0.5 nM Pol I or Klenow are due to a trace polymerase contamination in the β159 preparation. Alternatively, replication utilizing either the same M13 ssDNA template lacking the 5′ ³²P label (B and C) or a multiply nicked form of pBluescript KS II dsDNA (D and E), together with [³H]dTTP-dNTPs, was quantified by liquid scintillation spectroscopy of acid-insoluble products collected on 2.4-cm glass fiber filters as described in Materials and Methods. Reaction mixtures contained the indicated amounts (0.05, 0.5, or 5 nM) of Pol I (B and D) or the Klenow fragment of Pol I (C and E).