Figure 3.

Junction-specific cleavage by RNase HI is independent of any RNA/DNA heteroduplex. Above the appropriate lanes, substrate structure representations, and reaction times (min) are given. Gray lines indicate RNA and solid lines depict DNA. Reactions were performed as described in Fig. 2. (A) A 5′-radiolabeled oligonucleotide of (³²P)RNA_13nt⋅DNA_38nt was annealed to a DNA template complementary to only the DNA sequence (5′-TACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA-3′). Over time, RNase HI activity released an RNA_12nt product. (B) RNase HI activity also was examined on the RNA_13nt⋅DNA_38nt oligonucleotide in the absence of any DNA template. Again, a specific RNA_12nt segment was released. (C) RNase HI activity on a fully annealed substrate generated the RNA_12nt product at the same rate as in A and B. The size marker (M) containing an RNA_13nt transcript is indicated.