the target protein were dialysed twice against 5 l of 10-mM potassium phosphate and 5-mM EDTA (pH 7.4; buﬀer C). The dialysed solution was diluted to 50 ml with buﬀer C and loaded onto a hydroxyapatite column, BioScale CHT5-I (bed volume of 20 ml; Bio-Rad Laboratories Inc., Hercules, CA, USA) equilibrated with buﬀer C. The ﬂow-through fraction was collected. The proteins were eluted with a linear gradient of 10– 500 mM K3PO4 (pH 7.4) and 5-mM EDTA. Puriﬁed TTHB178 was concentrated to 10 mg/ml in 20-mM Tris– HCl, 100-mM KCl and 60% glycerol (pH 8.0) and stored at 20 C. Peptide mass ﬁngerprinting (33) conﬁrmed that the puriﬁed protein was TTHB178. Size-exclusion chromatography Size-exclusion chromatography was performed 25 C by using a Superdex 75 HR column (1 30 cm; GE Healthcare Biosciences) in an A¨KTA system (GE Healthcare Biosciences). The 100 ml of puriﬁed TTHB178 (0.75 mg/ml) was loaded onto the column and eluted at a ﬂow rate of 0.5 ml/min with 20-mM Tris–HCl and 100-mM KCl (pH 8.0). The elution proﬁle was monitored by recording the absorbance at 280 nm. The column was calibrated using apoferritin (443 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), thyroglobu- lin (66.9 kDa) and cytochrome c (12.4 kDa). Dynamic light scattering experiment The 2.0 mg/ml of TTHB178 was prepared in 20-mM Tris– HCl and 100-mM KCl, pH 7.5, and was passed through 0.02 mm Whatman Anodisc 13 Supported Membrane Filter. The 12 ml of the protein solution was loaded into a quartz cuvette and then analysed by dynamic light scat- tering instrument, DynaPro MSXTC/12/F with a gallium– arsenite diode laser, DynaPro-99-E-50 (Protein Solutions Inc., Charlottesville, VA, USA) at 20 C. The data were analysed using the Dynamics version 6.3.18 (Proteins Solutions Inc., Charlottesville, VA, USA). The sample was analysed a minimum of 10 times and the resulting data were analysed to estimate apparent molecular weight assuming a globular protein in an aqueous solution. The hydrodynamic radius (Rh) value was calculated with the Stokes–Einstein equation (Equation 1) using the obtained translational diﬀusion coeﬃcient (DT): Rh ¼ kBT=6 DT where kB is the Boltzmann constant, T the absolute tem- perature, Z the solvent viscosity and Rh the hydrodynamic radius. Molecular mass of the protein in the solution was estimated from Rh using an empirical curve of known proteins (Equation 2). Molecular mass ¼ 3366:5 R2:3398 h 1 ð Þ 2 ð Þ Mass analysis by using Fourier transform ion cyclotron mass spectrometer The products of the exonuclease reaction were analysed by Fourier transform ion cyclotron mass spectrometer (FT-ICR MS) with electrospray ionization. In brief, Nucleic Acids Research, 2010, Vol. 38, No. 17 5695 21-mer ssDNA (21f) was reacted with 3-mM TTHB178 in 20-mM Hepes–KOH, 100-mM KCl and 5-mM MgCl2, pH 7.5, at 37 C for 0, 1, 5 and 10 min, respectively. Each reactant was mixed with ion-pairing agent, butyl dimethyl ammonium carbonate, pH 8.0 to a ﬁnal 25-mM concentration. The mixture was loaded onto a self-made reverse-phase column using C18 Empore disk (3M Co., St Paul, MN, USA), after equilibration with 25-mM butyl dimethyl ammonium carbonate. The column was further washed with 5% acetonitrile contain- ing 25-mM butyl dimethyl ammonium carbonate and then the products were eluted with 50% acetonitrile. Basic additives, piperidine (pH 9) and imidazole (pH 8) for mass analysis of nucleic acids under negative mode were added to each eluent to a ﬁnal concentration of 25 mM, respectively. The resulting solution was subjected to an APEX IV, FT-ICR MS shielded with 9.4 T magnet (Bru¨ ker Daltonics Inc., MA, USA), by electrospray ion- ization under 2 ml/min ﬂow rate as describe in a previous report (34). Exonuclease assays Single-stranded oligonucleotides were synthesized (BEX Co., Tokyo, Japan) and their 50-termini were radiolabelled with [g-32P]ATP using T4 polynucleotide kinase (Takara Bio, Shiga, Japan) at 37 C for 1 h. The substrates with 30-overhanging, Y and gapped ﬂap structures were yielded by hybridizing 50sf with 40sr, 50sf with 28sr and 50sf with 28sr and 21sr (Table 2), respectively. In the case of 30-end labelling, an oligonucleotide (Table 1; 21r; 50-GG GTGTTGCTTTAGTTGTCAT-30) was radiolabelled with [a-32P]cordycepin-50-triphosphate (PerkinElmer Life & Analytical Sciences, Boston, MA, USA) by using terminal deoxynucleotidyl transferase (Promega Co., Fitchburg, WI, USA). The radiolabelled substrates were incubated with 3-mM TTHB178 in 20-mM Hepes–KOH, 100-mM KCl and 5-mM MgCl2 (pH 7.5). The total reaction volume was 10 ml. The reaction temperatures and times were as indicated in the ﬁgure legends. The re- actions were stopped by the addition of an equal volume of phenol, CHCl3 and isoamyl alcohol (25:24:1) as well as 1 ml of 100-mM EDTA. The mixture was centrifuged at 15 000g at 4 C for 10 min and the aqueous phase was mixed with an equal volume of sample buﬀer (5-mM EDTA, 80% deionized formamide, 10-mM NaOH, 0.1% bromophenol blue and 0.1% xylene cyanol). The samples were analysed by electrophoresis through denaturing 25% polyacrylamide gels with 1 TBE buﬀer (89-mM Tris–borate and 2-mM EDTA), and the gels were dried and placed in contact with an imaging plate. The substrates and products were detected and analysed with a BAS2500 image analyzer (Fuji Photo Film, Tokyo, Japan). In the kinetic analysis, 10-nM radiolabelled substrates were mixed with 0.1, 1, 5, 10, 50, 100, 300, 500 or 1000-mM of non-labelled substrates and then reacted with 100-nM TTHB178 for 30 min at 37 C or 5 min at 60 C, respectively. The values of the initial rates were calculated based on the amount of undegraded substrates according to a previously described