Figure 4
RNase H activity and binding affinities of mutant RNase H (D599N-H724N). (A) Qualitative RNase H assay. The 10/10-mer RNA/DNA substrate, 5’ labeled at the RNA strand with 32P, is depicted on top of the figure. Cleavage products obtained with 670 nM substrate and 20 μM wt RNase H (RHwt) or mutant RNase H-(D599N-H724N) (RH_D599N_H724N) were separated on a 15 % sequencing gel and visualized by phosphoimaging. Arrows and numbers indicate the cleavage sites identified on the gel. The first nucleotide of the RNA hybridized to the 3’ OH nucleotide of the DNA strand is designated −1. The incubation times are shown on top of the gel. C, control assays were performed in the absence of enzyme for 0 minute, 60 minutes and 6 hours. (B) Determination of KD-values by fluorescence anisotropy measurements. 25 nM of the 10/10-mer RNA/5’-DY647-DNA substrate were titrated with increasing amounts of wt RNase H (open circles) or mutant RNase H-(D599N-H724N) (closed circles). The fit of the curve to the data [5] resulted in KD-values of 23 μM (± 3) for the wt, and 31 μM (± 5) for the mutant RNase H-(D599N-H724N) enzyme.