might be involved in the downstream reaction. Third, the role of ssExos is completely uncertain in the repair of other damages, such as oxidized bases, methylated bases or abasic sites in DNA. Thermus thermophilus HB8 has a small genome size, 2.2 Mb, and an extremely high optimum growth tem- perature, 75 C (18). Proteins from this eubacterial strain are extremely stable and suitable for in vitro characteriza- tion. Therefore, we selected T. thermophilus HB8 for sys- tematic study of the structures and functions of all proteins from a single organism (18). We have already investigated many DNA repair enzymes from the strain (9,19–21), including a 50–30 ssExo, RecJ. It seemed that T. thermophilus HB8 possesses a 30–50 ssExo, TTHB178, in addition to RecJ. We found the DnaQ exonuclease motif in the N-terminal region of TTHB178 (Figure 1) that had been annotated as a functionally unknown protein. Most of the exonuclease domains of 30–50 ssExos are categorized into the DnaQ superfamily (22,23). The 30–50 exonuclease domains of the DnaQ superfamily share three conserved exonuclease motifs (Exo I–III) containing negatively charged amino acid residues (24), which coordinate two divalent metal ions to catalyse the phosphodiester bond cleavage (25,26). Recent reports have described the expres- sion of the tthb178 gene under the control of a transcrip- tional regulator, cyclic AMP-receptor protein (CRP), in T. thermophilus HB8 (27), implying that tthb178 plays a certain kind of biological role in the cell. As there seems to be no other candidate for the ssExos in T. thermophilus HB8 except for the proofreading domains of DNA polymerases, this organism is expected to have a single set of 30–50 ssExo (TTHB178) and 50–30 ssExo (RecJ). In majority of the organisms, including E. coli, yeast and humans, redundancy of the same polarity of ssExos makes it diﬃcult to clarify the rela- tionship between their cleavage polarities and their cellular functions (Supplementary Table S1). In contrast, Nucleic Acids Research, 2010, Vol. 38, No. 17 5693 T. thermophilus HB8 is suitable for investigating the dif- ference in cellular functions between 30–50 and 50–30 ssExos. Although we cannot exclude the possibility that T. thermophilus HB8 has additional ssExo with sequences diﬀerent from known enzymes, gene disruption of an ssExo is expected to aﬀect the phenotypes of the disrup- tion mutants directly. In this study, we investigated the phenotypes of the dis- ruption mutants of the tthb178 and recJ genes under DNA-damaging conditions. We also prepared the tthb178 gene product and examined its biochemical activity against various types of DNA in vitro. The results suggest that TTHB178 is a 30–50 ssExo that func- tions in several DNA repair pathways not only coopera- tively with but also independently of RecJ. MATERIALS AND METHODS Transcription analysis of tthb178 Thermus thermophilus HB8 cells were cultured overnight in TT broth (0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4-mM MgCl2 and 0.4-mM CaCl2; pH 7.2) at 70 C, diluted 100-fold in fresh TT broth and the diluted culture was incubated at 70 C. At each time point, cells were harvested by centrifugation at 2300g for 10 min at 4 C and stored at 20 C. Puriﬁcation of mRNA was carried out by using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol (28). The cDNA was synthesized by reverse tran- scription–PCR using forward primer 50-ACCTCTACGC CTTCCTCCTC-30 and reverse primer 50-CTCCTTGATT CTCTGGGCGG-30. The ampliﬁed fragment was 332 bp. Disruptions of tthb178 and recJ The gene mutants of T. thermophilus HB8 were con- structed by using a previously reported procedure (29). Figure 1. Amino acid sequence alignments of TTHB178 and the proteins belonging to the DnaQ superfamily. (A) Exonuclease motifs I, II and III of TTHB178 and exonucleases belonging to the DnaQ superfamily. The numbers to the left of the motifs indicate the distances from the protein N-termini. The predicted active site residues are highlighted in dark grey. (B) Schematic diagrams of TTHB178 and the other DnaQ superfamily proteins. DnaQ exo, ExonucX-T_C and POLBc epsilon mean the exonuclease domain of the DnaQ superfamily, the SH3-like and helical domains of E. coli EXOI and the DNA polymerase domain of type-B family DNA polymerases, respectively. TTH_TTHB178, T. thermophilus HB8 TTHB178; ECO_EXOI, E. coli ExoI; ECO_EXOX, E. coli ExoX; HSA_DPOE, Homo sapiens DNA polymerase e; BT4_DPOL, bacteriophage T4 DNA polymerase.