540 Nucleic Acids Research, 2011, Vol. 39, No. 2 Figure 3. Cleavage activity of Tma endo V on 50-labeled single-stranded and short I-containing substrates. (A) Exonuclease activity of Tma endo V mutants on 50-labeled single stranded I-containing substrate. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. (B) Exonuclease activity of Tma endo V mutants on 50-labeled short I-containing substrate. Cleavage reactions were performed as described in ‘Materials and Methods’ section with 100 nM enzyme and 10 nM substrate. incubation time, the intensities of the fragments also increased. After 1 min incubation, the high molecular weight fragments started to disappear, while the bands of low molecular weight started to intensify. These results indicated that Tma endo V possesses exonuclease activity that initiates non-speciﬁc degradation from the 50-end. The apparent rate constant of the non-speciﬁc 50-exonuclease activity was 0.55 per min (Figure 8B). The caveat is that although the apparent rate constant may be dominated by the 50-exonuclease activity, contribution from some non-speciﬁc endonuclease activity cannot be ruled out. DISCUSSION Endonuclease V is a versatile DNA repair enzyme with multiple nuclease activities on different DNA substrates, including deaminated bases and mismatched base pairs. Previous studies have indicated that endo V possesses multiple non-speciﬁc nuclease activities in addition to the inosine-speciﬁc endonuclease activity that nicks the inosine-containing strand at the 1 nt downstream of the lesion from the 30-side (3,4,16–18). However, the nature of the multiple nuclease activities is not deﬁned. This study takes advantage of the site-directed mutant repertoire we accumulated previously and uses different end labeled and internally labeled oligonucleotide substrates to investigate the nuclease activities. The biochemical data presented here allows us to dissect the nature of the multiple nuclease activities. Inosine-dependent endonuclease activity Endo V is known as a deaminated base repair enzyme. The inosine-dependent endonuclease activity is well docu- mented and is a relevant enzymatic activity in vivo (1,3–5,8,9,11,21,22). The inosine-dependent endonuclease activity is the primary enzymatic activity that is well main- tained within the protein structure (13,14). This is evidenced by the retention of the activity by most of the Tma endo V mutants tested except three active site mutants (D43A, D43C, D43H) (Figures 1 and 3). The inosine-dependent endonuclease activity allows endo V to initiate the removal of inosine in DNA. Inosine-dependent 30-exonuclease activity Previously, we detected 30-exonuclease activity from Tma endo V using binding defective mutants (13). Here we establish that the 30-exonuclease activity is inosine-dependent. Support for this notion primarily comes from biochemical analysis of some active site