Figure 4.

Stability of gene 4 primase and polymerase complex. T7 gene 4 primase was allowed to synthesize a tetraribonucleotide primer, 5′-pppACCC-3′, on a synthetic oligonucleotide template, 5′-CAGTCACGGGTCGTTTATCGTC-3′, containing the primase recognition sequence 5′-GGGTC-3′ (underlined). T7 DNA polymerase extends this oligoribonucleotide to the fourth nucleotide, at which point the extension terminates because of the incorporation of ddAMP at the fourth nucleotide. Using this strategy, a complex of gene 4 helicase/primase, ³²P-labeled primer–template, and DNA polymerase was formed as described in Materials and Methods. The protection of the labeled primer by the helicase/primase–polymerase complex from exonuclease III digestion was measured at different time points as indicated. The products of exonuclease III digestion were analyzed by 20% polyacrylamide-urea gel electrophoresis. An autoradiogram of the gel is shown above.