538 Nucleic Acids Research, 2011, Vol. 39, No. 2 RESULTS Cleavage of 50-labeled inosine- and non-inosine-containing substrates Endonuclease V is an authentic nuclease involved in repair of deaminated base damage such as inosine. The thermo- stable ortholog from Thermotoga maritima has been a valuable model system for biochemical investigation given its thermostability and ease of generating site-directed mutants. Previously, we have conducted a systematic site-directed mutagenesis of Tma endo V and generated over 60 site-directed mutants (13). Using the mutants with relatively low binding afﬁnity to inosine-containing DNA, we observed 30-exonuclease activity of Tma endo V in the presence of Mn2+ metal cofactor. However, the biochemical properties of the exo- nuclease activity were not deﬁned. This study attempts to understand the exonuclease activity in greater detail using 36 mutants and the wild-type (wt) Tma endo V (Figure 1A). Among them, D43, E89, D110 and H214 are part of the active site that coordinates metal binding and catalysis (14). We initially tested the nuclease activity using a 50 ﬂuorescently labeled inosine-containing substrate (Figure 1B). The inosine was placed at the 11th position from the 30-side so that endo V cleavage would generate a 51-mer, since it cuts at the 30-side 1 nt from inosine. If a mutant exhibited 30-exonuclease activity, a long ladder below the 51-mer was anticipated. Under the assay condition in which Mn2+ was used as a metal cofactor, all the mutants except D43A, D43C and D43H showed a strong 51-mer band, resulting from the speciﬁc inosine-dependent endo- nuclease activity (Figure 1B). The lack of activity in the D43 mutants ruled out the possibility that the observed activity was due to nuclease contamination from the expression host. Due to non-speciﬁc nuclease activities, the wt enzyme cleaved the speciﬁc 51-mer to small low molecular weight products (Figure 1B). Similar to the previous observation (13), some mutants produced a ladder that was indicative of 30-exonuclease activity (Figure 1B). The active site mutants in general did not show the 30-exonuclease activity except E89D, H214C, H214D and H214E (Figure 1B). Some mutants such as G113V, G121V and G136V previously known for their relatively low activity also showed minimal ladder forma- tion (13). Consistent with the substantial reduction of binding afﬁnity to inosine-containing DNA, H116E and in particular H116Y had little exonuclease activity (Figure 1B). Overall, the uniform ladders generated from some of the mutants indicated that Tma endo V possesses 30-exonuclease activity. To further conﬁrm the 30-exonuclease activity, we assayed DNA cleavage using a synthesized nicked inosine-containing DNA with (Figure 2A, nicked T/I sub- strate) or without the 30-downstream complementary DNA (Figure 2B, overhang T/I substrate). Regardless of which substrate tested, they all showed a very similar lad- dering pattern, suggesting that the 30-exonuclease activity did not depend on the 30-downstream complementary sequence. We then tested whether the same pattern would hold for single-stranded inosine-containing substrates. Indeed, the wt enzyme and the mutants all showed a similar laddering pattern with the full-length and the short single-stranded inosine-containing sub- strates (Figure 3). Since all the substrates tested so far contained a single inosine in the sequences, we set out to determine how the wt enzyme and mutants cleave non-inosine-containing DNA. Using a double-stranded substrate in which the T/I base pair was replaced with a T/A base pair, we measured the non-speciﬁc nuclease activities. As expected, the wt Tma endo V degraded the substrate to low molecular weight fragments due to non-speciﬁc activities [Figure 4A and (2)]. The majority of the binding defective mutants showed varying degrees of non-speciﬁc activities as indicated by the low molecular weight fragments near the bottom of the gel (Figure 4A, Q20A-R205K). The active site mutants exhibited little low molecular weight fragments (Figure 4A, D43A-H214E), suggesting that the non-speciﬁc activities were minimized. A very similar pattern was observed in the single-stranded non-inosine-containing substrate (Figure 4B). A signiﬁ- cant difference of the 30-exonuclease pattern between the inosine-containing and non-inosine-containing substrates was noted in some active site mutants (Figures 1 and 4). In particular, H214D mutant generated the well-formed ladder only with inosine-containing DNA, not with non-inosine-containing DNA. This result indicates that the 30-exonuclease activity requires the presence of an inosine site in the DNA. The minimal non-speciﬁc activity exhibited by the H214D mutant allowed us to better characterize the 30-exonuclease activity without the interference of non-speciﬁc activity as seen in the wt enzyme. Taking advantage of this unique nuclease property, we studied the kinetics of the 30-exonuclease activity using the H214D mutant. At the initial time points, we observed signiﬁcant cleavage at the inosine site to generate the 51-mer fragment and a small amount of lower molecu- lar weight fragments on a T/I substrate (Figure 5A, 2–5 min). After that, a long ladder was produced by the 30-exonuclease cleavage (Figure 5A). The intensities of bands below 51-mer position were initially low but then became stronger as the reaction progressed to ap- proximately below 43-mer position (Figure 5A). To verify this kinetic property, we then performed a time course analysis using the nicked inosine-containing substrate. The cleavage of the nicked inosine-containing substrate essentially showed the same laddering pattern (Fig. 5B). These results indicated that the H214D mutant did not dissociate frequently from the DNA in the initial 30-exonuclease action. After removing 8-nt, the frequencies of dissociation increased, which resulted in an intensiﬁed ladder. The apparent rate constant of the inosine-dependent 30-exonuclease activity for the nicked inosine-containing substrate was 0.025 per min. Cleavage of internally labeled inosine-containing substrate To better understand the 30-exonuclease activity, we designed an internally labeled substrate in which the ﬂuor- escent group was moved from the 50-end to the 14th