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. 2004 Sep 30;32(17):5223–5230. doi: 10.1093/nar/gkh865

Figure 2.

Figure 2

Activity of the Sso DNA primase on homo-polymeric and bubble-containing DNA templates. Sso DNA primase assays (reaction volume: 20 μl) were carried out using radioactive nucleotides at 5 μM with a specific activity of 10–50 μCi/nmol, as described in Materials and Methods. Aliquots of the reaction products (6 μl) were run on 20% polyacrylamide/urea gels. Assays were carried out using the following templates: the C-Bubble substrate and poly(dC) (A); the T-Bubble substrate and poly(dT) (B); the oligonucleotides used to prepare the T-Bubble substrate (bottom and top strands), the T-Bubble substrate and the T-Bubble substrate with arms of 28 nt (T-Bubble-la; E). (C) Reaction products synthesized by Sso DNA primase on the T-Bubble substrate in standard conditions were subjected to the following treatments: no digestion (lane 1) or extensive digestion with Dnase-free Rnase A (lane 2) or with DNAse I plus Rnase A (lane 3). 5′-radiolabelled synthetic DNA oligonucleotides were run as reference (lane 4) and their length is indicated on the right side of the gel. (D) M.jannaschii DNA primase was assayed on the T-Bubble substrate and poly(dT), as described in Materials and Methods. Lanes 1 and 3 of the gels shown in A, B and D and lanes 1, 3, 5 and 7 of the gel shown in E were loaded with control reaction with no DNA template.