A novel single-stranded DNA-specific 3′–5′ exonuclease, Thermus thermophilus exonuclease I, is involved in several DNA repair pathways

Abstract

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5′–3′ and 3′–5′ ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3′–5′ ssExo, resulted in significant increase in the sensitivity to H2O2 and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5′–3′ ssExo, showed little effect on the sensitivity to H2O2, but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3′–5′ ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3′–5′ ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.

Resistance to UV Irradiation Caused by Inactivation ofnurAandherAGenes in Thermus thermophilus

“…In contrast, the ΔrecJ strain showed a higher mutation frequency than the WT (Fig. 2), as previously reported (26). These results suggest that NurA and HerA have no effect on prevention of mutations that could be detected in this assay.…”

“…In contrast, the ΔrecJ strain showed a lower maximum cell density in the stationary phase, a lower cell viability (Fig. 1), and a longer cell shape than the WT (data not shown), as previously reported (26). These results suggest that NurA and HerA have no effect on cell growth and viability.…”

“…The gene disruption of some DNA repair proteins is known to increase sensitivity to UV irradiation: the ΔrecJ mutant was more sensitive to UV than the WT ( Fig. 4) (26). It should be mentioned that the survival percentage of the ΔrecJ strain was nearly unchanged at higher dose levels (60 to 96 J · m Ϫ2 ).…”

“…In T. thermophilus, RecJ has been thought to be the only enzyme that can perform the processing of the 5= ends of DNA in HR because this bacterium has no recBCD and recQ homolog (1). However, the T. thermophilus RecJ gene can be disrupted (26), indicating that other nucleases are involved in the 5=-end processing. In fact, the recJ recBCD and recJ recD double mutants show lethality in Acinetobacter baylyi, which has no other homolog for an end resection-related exonuclease (27).…”

In Vivo, in Vitro, and X-ray Crystallographic Analyses Suggest the Involvement of an Uncharacterized Triose-phosphate Isomerase (TIM) Barrel Protein in Protection against Oxidative Stress

“…Disruptions of tthb071, tthb070, and uvrC-The tthb071, tthb070, and uvrC null mutants of T. thermophilus HB8 (⌬tthb071, ⌬tthb070, and ⌬uvrC) were generated by substituting the target gene with the thermostable kanamycin resistance gene, HTK (27), through homologous recombination as described previously (28,29). The plasmids for gene disruptions were derivatives of the pGEM-T Easy vector (Promega Co., Madison, WI), constructed by inserting HTK, flanked by ϳ500-bp upstream and downstream sequences of each gene.…”