Nucleic Acids Research, 2011, Vol. 39, No. 2 537 Figure 1. Cleavage activity of Tma endo V on 50-labeled full-length T/I substrate. (A) Conserved motifs of Tma endo V. Site-directed mutants used in this study were listed above the arrows. (B) Cleavage activity of wt and mutant Tma endo V on 50-labeled full-length T/I substrate. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. The 50-end of the bottom strand was labeled with the Fam ﬂuorophore. The T/I base pair was underlined. (2,3,7,13,14,19). In this study, we investigated endonucle- ase and exonuclease activities from Tma endo V using 50-labeled, 30-labeled and 50-internally labeled inosine- containing DNA. Using 50-labeled T/I and T/A substrates and 50-internally labeled T/I substrate, we detected inosine-dependent 30-exonuclease activity. Using a H214D mutant that primarily retained inosine-dependent endonuclease and 30-exonuclease activity, we deﬁned the kinetic property of the inosine-dependent 30-exonuclease activity. Using 30-labeled T/I and T/A substrates, we detected and deﬁned non-speciﬁc 50-exonuclease activity. MATERIALS AND METHODS Reagents, media and strains All routine chemical reagents were purchased from Sigma Chemicals (St. Louis, MO, USA), Fisher Scientiﬁc (Suwanee, GA, USA) or VWR (Suwanee). Deoxyoligo- nucleotides were ordered from Integrated DNA Technologies Inc. (Coralville, IA, USA). LB medium was prepared according to standard recipes. GeneScan Stop Buffer consisted of 80% formamide (Amresco, Solon, OH, USA), 50 mM EDTA (pH 8.0) and 1% blue dextran (Sigma Chemicals). TB buffer (1 ) consisted of 89 mM Tris base and 89 mM boric acid. TE buffer con- sisted of 10 mM Tris–HCl, pH 8.0 and 1 mM EDTA. Escherichia coli host strain AK53 (mrrB , MM294) was from our laboratory collection (20). Tma endo V mutant proteins were puriﬁed as previously described (7,13). Oligonucleotide substrates The sequence of the 6-carboxyﬂuorescein (Fam)-labeld oligonucleotide is shown in Figure 1B. Oligonucleotides containing inosine were ordered from IDT and puriﬁed by polyacrylamide gel electrophoresis. The oligonucleo- tides were dissolved in TE buffer at a ﬁnal concentration of 10 mM. The two complementary strands with the un- labeled strand in 1.2-fold molar excess was mixed and incubated at 85 C for 3 min and allowed to form duplex DNA substrates at room temperature for >30 min. Tma endo V cleavage assays The cleavage reaction mixtures (10 ml) containing 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N0- [2-ethanesulfonic acid])–KOH (pH 7.4), 1 mM dithiothreitol, 2% glycerol, 5 mM MnCl2, 10 nM oligo- nucleotide DNA substrate and 100 nM Tma endo V protein were incubated at 65 C for 30 min. The reactions were terminated by adding an equal volume of GeneScan stop buffer. The reaction mixtures were then heated at 94 C for 3 min and cooled on ice. Samples (3.6 ml) were loaded onto a 16% denaturing polyacrylamide gel con- taining 7 M urea. Electrophoresis was conducted at 1500 V for 3.2 h using an ABI 377 sequencer (Applied Biosystems). Cleavage products and remaining substrates were visualized using GeneScan analysis software version 3.0. All experiments were repeated independently at least once. Some ﬁgures were assembled from multiple gel images.