Figure 3.

Endonuclease mutants can conduct the TPRT reaction on prenicked DNA substrates. (A) Schematic diagram summarizing the substrates and products of the TPRT reactions. The two strands of the DNA substrate are indicated by thin lines; the cDNA synthesized during the reaction is indicated with a thicker line and the RNA template is indicated with a wavy line. (B) Autoradiographs of the reaction products with the PA..D mutation (Left) and PE..D mutation (Right) separated on 1% agarose gels. Only plasmids that have undergone the TPRT reaction can be seen in these autoradiograms. For each reaction, 0.25 μg supercoiled or prenicked plasmid DNA was incubated with the R2 protein as in Fig. 2, except that 2 μCi [α-³²P]dATP was added to each reaction. Lanes: 1 and 5, no protein controls; 2–4, 4, 8, and 16 ng of the mutant protein incubated with the supercoiled target; 6–8, 4, 8, and 16 ng of the mutant protein incubated with the prenicked target; and 9, 16 ng of wild-type protein incubated with supercoiled DNA.