Figure 3.

Structural requirements of substrates for cleavage by TaqN and TaqNP. (A) Effect of the substrate duplex length. Upper shows suggested secondary structures of representative substrates complexed with various primers, in which the substrate duplexes contain either 9 (D9) or 6 (D6) base pairs. Incubation of these substrates TaqNP was as in Fig. 2 (but for 4 min), and the products are shown Lower. Arrows in the structures indicate deduced cleavage sites. (B) Requirement for a 3′ arm of the substrate duplex. The top of the figure shows suggested secondary structures of substrates made from a 5′ ³²P-labeled 206-nt fragment and oligonucleotides 30 or 42 nt long, which differ by the presence of a 12-nt self-complementary 3′ end. These substrates, formed by annealing of 2 nM 206-nt fragment and 100 nM 30-0 or 30-12 oligonucleotides, were incubated for 30 min at 55°C with either 50 ng of TaqNP or 300 ng of TaqN in 10 μl of 10 mM Tris⋅HCl, pH 8/100 mM KCl/1 mM MgCl₂/10 μg/ml tRNA. −, no enzyme. Arrows in the structures indicate deduced cleavage sites. (C) Requirement for a 5′ end of the substrate strand. Circular ssM13 mp19 DNA (50 ng) was incubated with 2.5 ng of TaqNP or 1 ng of TaqN in 10 mM Tris⋅HCl, pH 8/50 mM KCl buffer in the presence of 1 mM MgCl₂ (lanes 2 and 4) or 1 mM MnCl₂ (lanes 1, 3, and 5) at 55°C for 4 hours. The products were separated by electrophoresis in 1% agarose gel in 45 mM Tris⋅borate, pH 8.3/1 mM EDTA buffer.−, no enzyme.