Figure 5.

Oligonucleotide synthesis by wild-type primase and primases genetically altered at positions 31 and 33 on templates containing base substitutions for the cryptic cytosine. Oligonucleotide synthesis assays were performed with ATP and [α-³²P]CTP on synthetic ssDNA oligonucleotide templates containing primase recognition sites of sequence 5′-GCN-3′, where N is A, C, G, or T, as indicated (Fig. 5 A–C) or 5′-GCC^x-3′, where C^x is either C or ^4HC, as indicated (Fig. 5D). After synthesis, products were separated on a 25% denaturing PAGE gel and autoradiographed. Oligonucleotide products are identified. (A) Wild-type primase. (B) Primase H₃₃A. (C) Primases D₃₁A, D₃₁E, D₃₁N and D₃₁S. (D) Wild-type and D₃₁S primases (for comparison with D₃₁S, the products of wild-type primase were diluted 10-fold before electrophoresis).