Nucleic Acids Research, 2011, Vol. 39, No. 2 541 Figure 4. Cleavage activity of Tma endo V on 50-labeled non-inosine substrates. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. (A) Cleavage activity of wt and mutant Tma endo V on 50-labeled full-length T/A substrate. (B) Cleavage activity of wt and mutant Tma endo V on 50-labeled full-length single-stranded substrate. Figure 5. Time course analysis of cleavage activity by Tma endo V-H214D on 50-lableled full-length and nicked T/I substrates. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM H214D and 10 nM substrate. Reactions were stopped on ice at the indicated time points, followed by addition of an equal volume of GeneScan stop buffer. The sequences of the oligonucleotides are shown in Figure 1B. (A) Time course analysis of cleavage activity by H214D on 50-labeled full-length T/I substrate. (B) Time course analysis of cleavage activity by H214D on 50-labeled nicked T/I substrate. mutants. H214C, H214D and H214E failed to show any ladder on the 50-labeled non-inosine substrate but formed a distinct ladder on all 50-labeled inosine-containing sub- strates (Figures 1–4), suggesting that the 30-exonuclease activity relies on the primary cut at the inosine site to initiate the secondary enzymatic action. The observation of DNA cleavage activity on the single-stranded inosine- containing substrate but not the non-inosine-containing