Fig 4.

RNase H activity of WT and mutant recombinant RT enzymes. (A) Graphic representation of the RNA-DNA (kim40R-kim32D) substrate duplex used to monitor the cleavage efficiency of mutant and WT RTs. The 40-mer RNA kim40R was labeled at its 5′ terminus with ³²P and annealed to 32-mer DNA oligonucleotide kim32D. The positions of RNase H cleavage relative to the 3′ terminus of the DNA primer are indicated by arrows. (B) RNase H activity was analyzed by monitoring substrate cleavage in time course experiments. The time points were 0.2, 0.5, 1, 2, 6, 8, 10, and 16 min. The lane labeled time point 0 shows the uncleaved substrate in a control reaction without RT enzyme. The positions of cleaved products relative to the 3′ terminus of the DNA primer are indicated on the left. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis. Each experiment was repeated at least twice and yielded similar results on each occasion. The figure shows a gel from a representative experiment.