542 Nucleic Acids Research, 2011, Vol. 39, No. 2 Q20A Y80A Y80H L85A G113V H116Y H116T R118K G121VA138I R205K D43C D43E E89H D110A D110H H214A H214D WT F46A Y80F G83V R88E H116E H116Q R118A K119R G136V S182I D43A D43H E89A E89D D110C D110E H214C H214E 60-mer 51-mer 14-mer Figure 6. Cleavage activity of Tma endo V on internally labeled T/I substrate. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. The thymidine at position 14 from 50-end of the bottom strand was labeled with the Fam ﬂuorophore. Figure 7. Cleavage activity of Tma endo V on 30-labeled full-length T/I and T/A substrates. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. The 30-end of the bottom strand was labeled with the Fam ﬂuorophore. (A) Cleavage activity of wt and mutant Tma endo V on 30-labeled full-length T/I substrate. (B) Cleavage activity of wt and mutant Tma endo V on the 30-labeled full-length T/A substrate. DNA indicates that the 30-exonuclease does not rely on a nick or a recessive end; rather, it depends on the existence of inosine (Figures 3 and 4B). The more interesting kinetic property of the inosine-dependent 30-exonuclease activity is revealed from the time course analysis using the H214D mutant that has minimal non-speciﬁc nuclease activity (Figure 5). The ladders generated by H214D have a very distinct feature, i.e. the laddering patterns show little change over time. For example, the pattern at 60 min is quite similar to that at 7 min (Figure 5B). If the ladders are generated by non-speciﬁc 30-exonuclease activity, one