Figure 6

RNase H activity of HIV-1 subtype B and C recombinant wild type RTs. (A) Graphic representation of the substrate RNA/DNA (kim40R/kim32D) duplex used to monitor the RNase H cleavage efficiency of both recombinant RTs. The 40-mer RNA kim40R was labeled at its 5'-terminus by ³²P and annealed to 32-mer DNA oligo kim32D. -1, -10 and -20 are used as markers to indicate the positions of cleavage sites relative to the 3' end of the DNA primer. (B) The RNA-DNA substrate was incubated with the recombinant subtype B and C RT enzymes in assay buffer as described in Materials and Methods. RNase H cleavage was initiated by the addition of MgCl₂ and analyzed by monitoring substrate cleavage in time-course experiments in the absence (left panel) or presence (right panel) of a heparin trap. The position of cleaved products is indicated on the left. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis.