5700 Nucleic Acids Research, 2010, Vol. 38, No. 17 Figure 6. Exonuclease activity of TTHB178 against various DNA structures. (A–C) The 30-overhanging (50sf+40sr) (A), Y structure (50sf+20sr) (B) and gapped ﬂap structure (50sf+21sr+28sr) (C) DNAs were reacted with 3-mM TTHB178 for various reaction periods. The reaction time is indicated at the top of the panels. Assays for the Y structure and gapped ﬂap structure were carried out at 20 C to stabilize the short dsDNA region of the substrates. As TTHB178 showed relatively weak activity at 20 C compared with that at 37 C or 60 C, the assays were performed for a prolonged reaction time. The assay for 30-overhanging DNA was carried out at 37 C. ‘C’ means the substrate incubated without TTHB178 for 27 h. (D) Activity for an ssDNA with a 30-H terminus. The substrate 21-mer ssDNA (21r) was 30-end-labelled with [a-32P]cordycepin-50-triphosphate and reacted with 3-mM TTHB178 at 37 C. The reaction time is indicated at the top of the panel. ‘C’ means the substrate incubated without TTHB178 for 30 min. In all the panels, the digested products were analysed by electrophoresis through denaturing 8 and 25% polyacrylamide gels. ‘M’ means the 40- (in A) and 19-mer (in B and C) of marker DNAs. The bands seen near the bottom of the gels in B and C might be experimental artefacts because they were not observed reproducibly. and named this protein as T. thermophilus exonuclease I (Tth ExoI). Tth ExoI exhibited relatively high KM value compared with other ssExos. It may be reasonable to suppress the non-speciﬁc ssDNA-binding activity of an ssExo until it is required. For example, ssExo activity of Neisseria meningitidis exonuclease VII is regulated by the inter- action between the large and small subunits in response to environmental signals (57). The ssDNA-binding activity of Tth ExoI might be enhanced by the interaction with other proteins such as single-stranded DNA-binding protein that has been reported to interact with Haemophilus inﬂuenzae RecJ (35), E. coli RecJ (58) and E. coli ExoI (59). The most striking result in this study is that tthb178 showed a phenotype associated with defects in DNA repair systems. tthb178 and recJ showed a higher spontaneous mutation rate than the WT (Figure 2B and D). DNA mismatches generated by errors of DNA repli- cation are the major source of spontaneous mutation (49). MMR is responsible for the repair of such mismatches and deﬁciency of MMR genes, such as mutS or mutL, results in a considerable increase in the frequency of spontaneous mutation (10,15). In majority of the organisms, the required exonuclease polarity for MMR is unknown, although exonuclease activity should be necessary for the removal of the mismatch-containing patch generated by the MutS/MutL complex. The spontaneous mutation rate of tthb178– recJ was much higher than that of tthb178 or recJ (Figure 2B). The severe eﬀect of the double disruption can be interpreted as follows: Tth ExoI and RecJ function in two parallel pathways during MMR. Consequently, we propose a model for MMR in T. thermophilus HB8. In this model, Tth ExoI and RecJ act as ssExos with opposite polarities, which excise the mismatch-containing DNA patch after the unwinding of dsDNA by DNA helicases (Figure 8A). The results of our in vitro experiments support the ability of Tth ExoI to cleave the liberated ssDNA regions in unwound dsDNAs (Figure 6B and C). Intriguingly, the disruption of tthb178 resulted in a great increase in the sensitivity to H2O2, whereas that of recJ did not (Figure 2D). In addition, tthb178– recJ showed similar H2O2 sensitivity as tthb178. H2O2- induced oxidative stress is known to cause base oxidation and base deamination (47). These results raise the possi- bility that Tth ExoI is involved in repair pathways for such damaged bases whereas RecJ is not. It is well-known that oxidized base such as 8-oxoguanine or deaminated base such as uracil is repaired through base excision repair pathway. In this repair system, the N-glycosidic bonds of the damaged bases are incised by speciﬁc DNA glycosylases (60–62) to yield mainly an abasic site, a 50-deoxyribose- 5-phosphate residue. However, the exonuclease assay in this study clearly showed that Tth ExoI cannot excise the abasic site-containing ssDNA. A previous study also showed that RecJ has no 50-deoxyribose-5-phosphatase activity (54). Thus, it is suggested that Tth ExoI and RecJ do not play a crucial role in the base excision repair pathway in T. thermophilus HB8. The deaminated bases are also repaired by the alterna- tive repair pathway that has not been completely revealed even in E. coli. In this repair pathway, endonuclease V is reported to nick the 30-side of the lesion such as uracil, xanthine and hypoxanthine (46,63,64). Thermus thermophilus HB8 also possesses a gene encoding endo- nuclease V (TTHA1347). Our biochemical study showed that Tth ExoI cleaves the uracil-, xanthine-, hypoxanthine- containing ssDNA. On the basis of our results and those of the previous studies, we propose a model of a repair pathway for those deaminated bases (Figure 8B). In this model, Tth ExoI starts the excision from the entry point introduced by endonuclease V. As endonuclease V nicks the 30-side of the lesion, the 30–50 ssExo but not the 50–30 ssExo is expected to be required for removing the lesion