Nucleic Acids Research, 2011, Vol. 39, No. 2 543 Figure 8. Time course analysis of cleavage activity by wt Tma endo V on 30-labeled full-length T/A substrate. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme 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. (A) Time course analysis of cleavage activity by wt Tma endo V on 30-labeled full-length T/A substrate. (B) Plot of time course analysis of cleavage activity by wt Tma endo V on 30-labeled full-length T/A substrate. The experimental errors were estimated to be <10%. The cleavage data were ﬁtted into a ﬁrst-order equation using DeltaGraph version 5 (SPSS Inc. and Red Rock Software Inc.). would expect to see an initial accumulation of high molecular weight products. Later on, the high molecular weight products would gradually disappear, which is accompanied by accumulation of low molecular weight products. This will be similar to the laddering pattern observed by the non-speciﬁc 50-exonuclease activity shown in Figure 8A. However, this kind of kinetic behavior did not occur. As such, these data indicate that the 30-exonuclease action only occurs once on an inosine-containing DNA. After the enzyme removes the inosine lesion and dissociates from the DNA, it has little chance to re-bind to the 30-side to reinitiate a non-speciﬁc 30-exonulcease action. As a result, an even laddering pattern is generated over time. Thus, the kinetic pattern reinforces the notion that Tma endo V contains inosine-dependent 30-exonuclease activity. An alternative explanation for the even laddering pattern throughout the time course is that it is caused by non-speciﬁc endonuclease action that allows the enzyme to randomly nick the DNA. We found this explanation implausible. First, H214D mutant did not show any lad- dering pattern on the non-inosine substrates (Figure 4). Second, if it had been caused by non-speciﬁc endonuclease action, we could have seen the ladder generated by H214D mutant extend below the 14-mer mark in the internally labeled T/I substrate due to the multiple cuts around the Fam position (Figure 6). Instead, the ladder stopped at the 14-mer position (Figure 6). Finally, if it had been caused by non-speciﬁc endonuclease, the substrates should have been degraded to low molecular weight products over time (Figure 5). Does the wt enzyme possess non-speciﬁc 30-exonuclease activity then? Although the data presented here may not give a deﬁnitive answer, we tend to believe the answer is no, based on the following two reasons. First, no obvious ladders were observed with 50-labeled non- speciﬁc substrates (Figure 4). Second, the time course data from the 30-labeled substrate does not show any low molecular weight fragments at the bottom of the gel (Figure 8A), which would indicate non-speciﬁc 30-exonulcease activity. 50-exonuclease activity It has been proposed that Tma or E. coli endo V enzymes contain 50-exonuclease activity (16–18). We measured the 50-exonuclease activity using 30-labeled substrates. A ladder formed by some mutants such as Q20A and R205K is a good indication of 50-exonuclease activity (Figure 7). The 50-exonuclease activity is non-speciﬁc since it does not depend on the existence of an inosine site (Figure 7). In addition, the generation of low molecu- lar weight products as shown in Figure 4 is also indicative of non-speciﬁc 50-exonuclease activity. A more deﬁnitive proof of the 50-exonuclease activity comes from the time course analysis of the 30-labeled substrate (Figure 8A). At the initial time points, the 50-exonuclease action produces a ladder consisting of higher molecular weight fragments (Figure 8A, 5–20 s). At the later time points, the continu- ous 50-exonuclease action results in the disappearance of the high molecular weight fragments with concurrent accumulation of low molecular weight fragments (Figure 8A, 5–20 min). On the other hand, a prominent non-speciﬁc endonuclease would result in generation of fragments of all sizes initially and later accumulation of low molecular weight fragments. Nuclease activities and inosine repair Since endo V cuts at the 30-side of the lesion, the inosine damage still remains in the DNA. Additional enzymatic actions are required for the removal of the damage. Based on the results obtained from an oligonucleotide-based study in E. coli, after nicking by the inosine-dependent endonuclease activity of E. coli endo V, 2-nt from 50-side and 3 nt from 30-side are removed to create a gap (12). The enzymes responsible for the removal of inosine and sub- sequent creation of the 5-nt gap in the repair process are as yet unidentiﬁed. However, the existence of inosine- dependent 30-exonuclease and non-speciﬁc 50-exonuclease activities raises the possibility that endo V, facilitated by other factor(s) than the Mn2+ used in the in vitro study, could potentially be involved in the creation of the short gap during the repair process. If this is the case, then endo V can latch onto DNA and search for an inosine site.