Nucleic Acids Research, 2011, Vol. 39, No. 2 539 Figure 2. Cleavage activity of Tma endo V on 50-labeled nicked and overhang T/I substrates. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. The sequences of the oligonucleotides are shown in Figure 1B. (A) Cleavage activity of wt and mutant Tma endo V on 50-labeled nicked T/I substrate. (B) Cleavage activity of wt and mutant Tma endo V on 50-labeled overhang T/I substrate (51-mer). position thymine (Figure 6). The overall cleavage pattern was similar to the 50-end labeled T/I substrates. All the mutants except D43A, D43C and D43H generated a 51-mer speciﬁc inosine-dependent cleavage product at the 51-mer position (Figure 6). The same set of binding and active site mutants showed a 30-exonuclease ladder. A signiﬁcant deviation from the cleavage of the internally labeled T/I substrate is that for many mutants, in particu- lar H214C, H214D and H214E, the ladder halted at around 14-mer position (Figure 6). The implication of these observations will be discussed later. Cleavage of 30-labeled inosine- and non-inosine-containing substrates To characterize any potential nuclease activities that initiate from the 50-side, we switched the ﬂuorescent label to the 30-side of the oligonucleotide substrate with the inosine placed at the 33th position from the 30-label (Figure 7A). As expected, the endonuclease cleavage at the inosine site generated the 31-mer product (Figure 7A). Again, the wt enzyme degraded the product to low mo- lecular weight fragments, as seen in the bottom of the gel. Many mutants, in particular Q20A, H116T, R118A and R205K, still showed non-speciﬁc activity, but only R118A and R205K showed more extensive degradation of the 31-mer. G113V, H116Y, R118K, K119R, G121V, G136V and S182I generated little low molecular weight fragments, indicating attenuated non-speciﬁc activity (Figure 7A). The active site mutants showed varying degrees of speciﬁc endonuclease cleavage at the inosine site but little non-speciﬁc activity. We then tested the nuclease activities on the 30-labeled non-inosine substrate. The wt enzyme exhibited non-speciﬁc activities to degrade the DNA to low molecu- lar weight fragments (Figure 7B). Consistent with the results from the inosine-containing substrate, Q20A, H116T, R118A and R205K showed stronger non-speciﬁc activity than other binding defective mutants (Figure 7B, Q20A-R205K). The active site mutants exhibited minimal non-speciﬁc activity (Figure 7B, D43A-H214E). To further understand the kinetic property of the non-speciﬁc activity, we performed a time course analysis on the 30-labeled non-inosine substrate. At the initial 5 s time point, we observed a small amount of high molecular weight fragments generated from degradation of the non-inosine substrate (Figure 8A). With increased