Nucleic Acids Research, 2010, Vol. 38, No. 17 5699 Figure 5. Analyses of TTHB178 exonuclease activity by using 50 radiolabelled substrates. (A) Substrate speciﬁcity of TTHB178 exonuclease activity. TTHB178 (3 mM) was incubated with 10-nM 50-end-labelled 21-mer ssDNA (21f), 21-bp dsDNA (21f+21r) or 21-mer ssRNA (21rna) in the presence of 5-mM Mg2+ at 37 C. The reaction time is shown at the top of the panels. (B) Dependence of the exonuclease activity on divalent metal ions. The 10-nM 50-end-labelled ssDNA (21f) was incubated with 3-mM TTHB178 at 37 C for 2 h. The reaction mixture contained 5 mM of the respective divalent metal ions. (C) Dependence of the exonuclease activity on the concentrations of Mg2+, Mn2+ and Co2+. The 10-nM 50-end-labelled ssDNA (21f) was incubated with 3-mM TTHB178 at 37 C for 2 h in the presence of various concentrations of divalent cations. The concentrations of the divalent metal ions are indicated at the top of the panels. Table 2. Steady-state kinetic parameters of the TTHB178 exonuclease activity for ssDNAs Length (mer) Temperature kcat ( C) (s 1) 0.71 ± 0.23 7.5 ± 0.023 0.40 ± 0.15 5.3 ± 2.10 0.23 ± 0.12 1.9 ± 0.48 0.17 ± 0.016 2.3 ± 0.10 0.19 ± 0.046 1.0 ± 0.41 KM (mM) 280 ± 83 480 ± 16 350 ± 31 440 ± 120 260 ± 105 290 ± 58 240 ± 45 320 ± 47 130 ± 52 160 ± 39 kcat/KM (s 1 M 1) 2.6 103 ± 0.19 16 103 ± 0.49 1.1 103 ± 0.49 12 103 ± 3.70 0.83 103 ± 0.20 7.0 103 ± 2.40 0.72 103 ± 0.066 7.5 103 ± 1.40 1.7 103 ± 0.58 6.4 103 ± 0.87 10 21 28 40 50 37 60 37 60 37 60 37 60 37 60 mimicked the intermediate structures, which were generated by the unwinding of nicked dsDNAs by a DNA helicase. Furthermore, TTHB178 hardly degraded the ssDNA whose 30-terminus was radiolabelled with [a-32P]cordycepin-50-monosphate (Figure 6D). The cordycepin-labelled ssDNA had 30-H instead of 30-OH at its 30-terminus. The 30-OH group of the substrate would be essential for the TTHB178 activity. We also examined the exonuclease activity for ssDNA substrates containing various kinds of damaged bases. As a result, TTHB178 degraded ssDNAs containing hypo- xanthine, xanthine, uracil, O4-methylguanine and O6-methylthymine (Figure 7A–D). At 37 C, the degrad- ation stopped at the positions of hypoxanthine and xanthine in the respective substrates, but the degradation proceeded beyond these non-canonical bases at 60 C. In contrast, only slight activity was observed even at 60 C when the substrate contained 8-oxoguanine or an abasic site (Figure 7E and F). Based on these results, it is con- sidered that TTHB178 can be involved in the excision step of the DNA repair pathways for deaminated and methylated bases. DISCUSSION Our study showed that TTHB178 possesses 30–50 exo- nuclease activity. This result supports the prediction that TTHB178 is a member of the DnaQ superfamily exonucle- ases. Detailed biochemical analyses revealed that TTHB178 has several characteristic features that are similar to or diﬀerent from those of E. coli ExoI and ExoX. First, gel-ﬁltration chromatography suggested that TTHB178 can form a homodimer in solution (Figure 3) unlike ExoI and ExoX. It is reported that human DnaQ superfamily ssExos, TREX1 and TREX2, also can exist as a dimer (50,51). The dimerization ability of TREX2 has been discussed in a model of allosteric eﬀect to ensure the cooperative binding to substrate DNA (52). It will be interesting to elucidate the signiﬁ- cance of dimerization in DNA-binding activity of TTHB178. Second, TTHB178 speciﬁcally degraded ssDNA, but not dsDNA and ssRNA (Figure 5A). Such a strict speciﬁcity is similar to that of E. coli ExoI and ExoX (37,38,53). Finally, TTHB178 did not degrade an abasic site-containing ssDNA (Figure 7E and F), indicating that TTHB178 does not possess DNA deoxyribophosphodiesterase activity as exhibited by E. coli ExoI (54–56). From these results, we concluded that TTHB178 is a novel ssDNA-speciﬁc 30–50 exonuclease