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 H(2)O(2) 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 H(2)O(2), 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.

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 ε; BT4_DPOL, bacteriophage T4 DNA polymerase.

Figure 2.

Effects of the disruptions of the tthb178 and recJ genes on the growth of T. thermophilus HB8. (A) Growth curves of the WT (circles), ΔrecJ (triangles), Δtthb178 (inverted triangles) and the double disruptant (squares). Growth was monitored by measuring the absorbance at 660 nm. (B) Spontaneous mutation rates of each strain to the streptomycin-resistant strain. (C) Sensitivity to 254-nm UV-C irradiation. The survival ratios are shown as a bar graph. (D) Sensitivity to H₂O₂. The survival ratios were plotted against the H₂O₂ concentration. The symbols are the same as in (A). In all panels, the data represent the averages of at least three independent experiments and each bar indicates the SD.

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 μg 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), β-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. R_h was calculated on the basis of the observed D_T as described in ‘Materials and Methods’ section.

Figure 4.

Analysis of the exonuclease activity by using FT-ICR MS. (A) The nomenclature scheme used for oligonucletide ions (72). The four possible cleavages are indicated by the lower case letters a, b, c and d for ions containing the 5′-OH group and w, x, y and z for ions containing the 3′-OH group. The numerical subscripts indicate the number of bases from the respective termini. FT-ICR MS can achieve the simultaneous identification of these ions. (B) The deconvoluted mass spectra of the product ssDNAs of TTHB178. The 21-mer ssDNA (21f) was reacted with 3-μM TTHB178 at 37°C. The product ssDNAs were purified as described in the ‘Materials and Methods’ section and then analysed by using FT-ICR MS. The reaction time is shown in the panel. (C) The measured and theoretical masses of each peak (named A to Q) are listed and the corresponding sequences are also shown as the ‘identified sequence’. The measured mass coincided with the theoretical mass ∼10-ppm mass measurement accuracy.

Figure 5.

Analyses of TTHB178 exonuclease activity by using 5′ radiolabelled substrates. (A) Substrate specificity of TTHB178 exonuclease activity. TTHB178 (3 μM) was incubated with 10-nM 5′-end-labelled 21-mer ssDNA (21f), 21-bp dsDNA (21f + 21r) or 21-mer ssRNA (21rna) in the presence of 5-mM Mg²⁺ at 37°C. The reaction time is shown at the top of the panels. (B) Dependence of the exonuclease activity on divalent metal ions. The 10-nM 5′-end-labelled ssDNA (21f) was incubated with 3-μM TTHB178 at 37°C for 2 h. The reaction mixture contained 5 mM of the respective divalent metal ions. (C) Dependence of the exonuclease activity on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺. The 10-nM 5′-end-labelled ssDNA (21f) was incubated with 3-μM TTHB178 at 37°C for 2 h in the presence of various concentrations of divalent cations. The concentrations of the divalent metal ions are indicated at the top of the panels.

Figure 6.

Exonuclease activity of TTHB178 against various DNA structures. (A–C) The 3′-overhanging (50sf + 40sr) (A), Y structure (50sf + 20sr) (B) and gapped flap structure (50sf + 21sr + 28sr) (C) DNAs were reacted with 3-μM 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 3′-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 3′-H terminus. The substrate 21-mer ssDNA (21r) was 3′-end-labelled with [α-³²P]cordycepin-5′-triphosphate and reacted with 3-μM 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.

Figure 7.

Excision assay for ssDNAs containing various kinds of damaged bases. The 5′-end-labelled ssDNA containing a damaged base was reacted with 3-μM TTHB178 at 37 or 60°C. The respective substrates contained hypoxanthine (A), xanthine (B), uracil (C), 8-oxoguanine (D), a reduced abasic site (E), an abasic site (F), O⁴-methylthymine (G) and O⁶-methylguanine (H). In all the panels, ‘C’ means the substrate incubated at 60°C for 60 min without TTHB178. ‘M’ means the 16- (in A and B), 10- (in C, D, E, G and H) and 9-mer (in F) marker DNAs. The reaction time is shown at the top of the panels.

Figure 8.

Proposed models of the DNA repair pathways in T. thermophilus HB8. (A) The model of MMR. A DNA mismatch is generated by misincorporation of a base during DNA replication. The MutS/MutL complex recognises the mismatch and nicks the 3′- and 5′-sides of the incorrect base to create a DNA patch for removal. DNA helicases, such as UvrD, and RecJ or ExoI excise the error-containing patch. DNA polymerase fills the gap to complete the repair. (B) The model of the repair pathway for deaminated bases. Reactive oxygen species, such as hydroxyl radicals, attack the base to yield deaminated bases. Endonuclease V recognises a deaminated base and hydrolyses the second phosphodiester bond of the 3′-side of the bases. A DNA helicase unwinds the chain, and then, ExoI digests the lesion-containing ssDNA. (C) The model of DSB repair. UV-C irradiation causes DSBs in DNA. RecJ processes the termini to the 3′-overhanging structure in cooperation with a DNA helicase. The homologous pairing and re-synthesis of the DNA strand yield Holliday junctions. The resolution of Holliday junctions completes the repair.