death phases, unlike in the case of the WT (data not shown). These results suggested that T. thermophilus HB8 requires TTHB178 as well as RecJ for optimal growth under conditions without external DNA- damaging stress. To examine the possible involvement of TTHB178 and RecJ in DNA repair processes in T. thermophilus HB8, we ﬁrst measured the spontaneous mutation rate of the disruptants to a streptomycin-resistant strain (39). The spontaneous mutation rates of tthb178 and recJ were approximately four-fold and three-fold higher than that of the WT, respectively (Figure 2B). Interestingly, tthb178– recJ showed a signiﬁcantly higher rate than the single-disruption cells (Figure 2B). The streptomycin- resistant strains obtained here must have mutations within the rRNA gene (39,40) and such spontaneous mu- tagenesis can be accelerated by defects in several DNA repair systems such as MMR. Therefore, the observed increase in mutation frequency suggested that both TTHB178 and RecJ are involved in DNA repair. We then examined the growth phenotypes of these disruptants under DNA-damaging conditions. The dis- ruption of tthb178 did not aﬀect the sensitivity to UV-C irradiation at 254 nm (Figure 2C). On the other hand, recJ exhibited three-fold higher sensitivity to UV-C than the WT and tthb178 (Figure 2C). The major damages caused by UV-C irradiation are cyclobutane pyr- imidine dimers, pyrimidine-pyrimidone (6-4) photoprod- ucts and DSBs (41–45). The observed increase in the sensitivity indicated that RecJ is intimately involved in the repair of these lesions whereas TTHB178 is not. Nevertheless, it should be noted that the survival ratio of UV-C-irradiated tthb178– recJ was lower than that of DrecJ (Figure 2C). This result raised the possibility that TTHB178 also participates in the repair pathway for those lesions in the cell strain lacking the recJ gene product. In contrast, the disruption of tthb178 caused drastic increase in the sensitivity to H2O2, whereas the disruption of recJ did not (Figure 2D). In addition, tthb178–DrecJ exhibited a similar survival ratio to tthb178. Reactive oxygen species generated from H2O2 are responsible for Nucleic Acids Research, 2010, Vol. 38, No. 17 5697 the oxidation and deamination of bases, which result in transversion and transition mutations, respectively (46,47). Therefore, the increased sensitivity of tthb178 to H2O2 suggested that TTHB178 is involved in the repair pathway for such damaged bases. Thus, our in vivo experiments indicated that the tthb178 and recJ genes are required for several DNA repair pathways in T. thermophilus HB8. Exonuclease activity of TTHB178 In order to characterize TTHB178 biochemically, we overexpressed TTHB178 in E. coli and puriﬁed it to homogeneity (Figure 3A). Size-exclusion chromatography was performed to examine the self-association ability of TTHB178. The result showed that TTHB178 was eluted with a single peak corresponding to an apparent molecular mass of 62 kDa (Figure 3B). As the molecular mass of TTHB178 was calculated to be 33 kDa according to its amino acid sequence, this result implied that TTHB178 exists in a dimeric state in solution. The observed shoulder of the main peak might represent the slight tendency of TTHB178 to form a larger complex in the solution. Dynamic light scattering experiment was also performed to evaluate the dimerization ability of TTHB178. The measurement gave an Rh value of 3.6 nm, suggesting that the molecular mass of the particle in the TTHB178 solution is about 66 kDa (Figure 3C). Thus, the result of dynamic light scattering experiment also supports the dimerization of TTHB178. To test the prediction that TTHB178 has 30–50 exonucle- ase activity, the activity was examined using ssDNA as a substrate. The ssDNA that reacted with TTHB178 was subjected to FT-ICR MS analysis. FT-ICR MS is a powerful tool for the characterization of nuclease activity as it achieves precise and simultaneous identiﬁca- tion of the length, nucleotide content, and nature of the 50-termini and 30-termini of all products (Figure 4A). As the reaction time increased, the shorter products became obvious and all of them were the b-series ions that lack the 30-terminal region of the substrate DNA (Figure 4B and C). This result strongly indicated that Figure 3. Preparation of recombinant TTHB178. (A) Recombinant TTHB178 was puriﬁed as described in the ‘Materials and Methods’ section and then subjected to SDS–PAGE. The 3 mg of protein was loaded on the gel. The calculated molecular mass of TTHB178 is 33 kDa. The arrow indicates the band of TTHB178. (B) Size-exclusion chromatography. TTHB178 (0.75 mg/ml) was loaded onto a Superdex 75 HR column. The apparent molecular mass of the main peak was estimated to be 62 kDa, from the calibration curve shown in the inset. Apoferritin (443 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), thyroglobulin (66.9 kDa) and cytochrome c (12.4 kDa) were used as molecular size markers. (C) Dynamic light scattering measurement. Rh was calculated on the basis of the observed DT as described in ‘Materials and Methods’ section.