5698 Nucleic Acids Research, 2010, Vol. 38, No. 17 Figure 4. Analysis of the exonuclease activity by using FT-ICR MS. (A) The nomenclature scheme used for oligonucletide ions (72). The four possible cleavages are indicated by the lower case letters a, b, c and d for ions containing the 50-OH group and w, x, y and z for ions containing the 30-OH group. The numerical subscripts indicate the number of bases from the respective termini. FT-ICR MS can achieve the simultaneous identiﬁcation of these ions. (B) The deconvoluted mass spectra of the product ssDNAs of TTHB178. The 21-mer ssDNA (21f) was reacted with 3-mM TTHB178 at 37 C. The product ssDNAs were puriﬁed as described in the ‘Materials and Methods’ section and then analysed by using FT-ICR MS. The reaction time is shown in the panel. (C) The measured and theoretical masses of each peak (named A to Q) are listed and the corresponding sequences are also shown as the ‘identiﬁed sequence’. The measured mass coincided with the theoretical mass 10-ppm mass measurement accuracy. TTHB178 possesses exonuclease activity that degrades ssDNA from the 30-end to the 50-end and that TTHB178 hydrolyses a phosphodiester bond at the 30-side of the phosphate. The 30–50 exonuclease activity of TTHB178 was also conﬁrmed by electrophoretic analyses using 50-end-labelled ssDNAs as substrates. As shown in Figure 5A, TTHB178-digested products exhibited a ladder pattern of DNA fragments on the gel, which sug- gested that TTHB178 degrades ssDNA from the 30-end to the 50-end. We conﬁrmed that the elution proﬁle of the observed exonuclease activity from Superdex 75 HR column was exactly matched with that of TTHB178 (Supplementary Figure S5), indicating that the observed activity is derived from TTHB178. The results also showed that TTHB178 speciﬁcally cleaves an ssDNA (Figure 5A). No activity against ssRNA and dsDNA was observed in spite of the pro- longed reaction time (up to 4 h). It was also shown that the activity required divalent cations such as Mg2+, Mn2+ or Co2+ (Figure 5B and C). Our previous study showed that the intracellular concentrations of Mn (0.16 mM) and Co ions (not detected) are signiﬁcantly lower than that of Mg ions (35 mM) in T. thermophilus HB8 cells (48). The concentrations of Mn2+ and Co2+ were thought to be in- suﬃcient for the activation of TTHB178 exonuclease activity in vivo. Therefore, the assays for exonuclease activity were carried out in the presence of Mg2+. The steady-state kinetic parameters of the TTHB178 exonuclease activity were determined (Table 2) based on the reduction rate of undegraded substrates (21,35,36). The reaction temperature did not aﬀect the KM values of the respective substrates, but the kcat values at 60 C were higher than those at 37 C. As the substrates became shorter, the KM values became higher, showing the pref- erential binding of TTHB178 to longer ssDNAs. On the other hand, the kcat values of longer ssDNAs were lower than those of the shorter ones. Therefore, the kcat/KM values, the index of the eﬃciency of an enzyme, were not aﬀected by the lengths of the substrates. The kcat/ KM values were 4- to 10-fold higher at 60 C than at 37 C, which indicated that the observed digestion of ssDNA was certainly performed by a protein from a thermophile (i.e. T. thermophilus HB8) and not by a contaminated protein from a host cell. DNA structure and lesion speciﬁcity of the exonuclease activity We further tested the speciﬁcity of the TTHB178 exo- nuclease activity to the structure and lesion of substrate DNA. The 30-overhanging, Y and gapped ﬂap structures were used as substrates. These are possible intermediate DNA structures generated during the processes of several DNA repair pathways (49). The results indicated that TTHB178 can digest the ssDNA regions of the three sub- strates (Figure 6A–C). The Y and gapped ﬂap structures