Bax1 is a novel endonuclease: implications for archaeal nucleotide excision repair

Abstract

The helicases XPB and XPD are part of the TFIIH complex, which mediates transcription initiation as well as eukaryotic nucleotide excision repair (NER). Although there is no TFIIH complex present in archaea, most species contain both XPB and XPD and serve as a model for their eukaryotic homologs. Recently, a novel binding partner for XPB, Bax1 (binds archeal XPB), was identified in archaea. To gain insights into its role in NER, Bax1 from Thermoplasma acidophilum was characterized. We identified Bax1 as a novel Mg(2+)-dependent structure-specific endonuclease recognizing DNA containing a 3' overhang. Incision assays conducted with DNA substrates providing different lengths of the 3' overhang indicate that Bax1 specifically incises DNA in the single-stranded region of the 3' overhang 4-6 nucleotides to the single-stranded DNA/double-stranded DNA junction and thus is a structure-specific and not a sequence-specific endonuclease. In contrast, no incision was detected in the presence of a 5' overhang, double-stranded DNA, or DNA containing few unpaired nucleotides forming a bubble. Several Bax1 variants were generated based on multiple sequence alignments and examined with respect to their ability to perform the incision reaction. Residues Glu-124, Asp-132, Tyr-152, and Glu-155 show a dramatic reduction in incision activity, indicating a pivotal role in catalysis. Interestingly, Bax1 does not exhibit any incision activity in the presence of XPB, thus suggesting a role in NER in which the endonuclease activity is tightly regulated until the damage has been recognized and verified prior to the incision event.

FIGURE 1.

T. acidophilum Bax1 forms a complex with XPB. A, size exclusion chromatography was performed to verify the presence of a stable XPB-Bax1 complex. Bax1 was first analyzed individually (shown in black) followed by a 1:1 stoichiometric mixture of Bax1 and XPB (shown in gray). mAU = milli absorption units. Peak fractions were analyzed via SDS-PAGE to confirm complex formation. B, both Bax1 and the purified XPB-Bax1 complex were subjected to analytical ultracentrifugation in separate experiments to support the finding that XPB and Bax1 interact in solution (same color coding as in A).

FIGURE 2.

T. acidophilum Bax1 is an active structure-specific endonuclease. A, the DNA substrate NDT/NDB22 (shown in C) was labeled at the 5′ end of the upper strand and used at a concentration of 10 nm. Arrows indicate full-length DNA as well as the incision product. Increasing concentrations of Bax1 lead to increasing amounts of the incision product. B, Bax1 incises 4–6 nt away from a dsDNA/ssDNA junction. Incision assays employing diverse DNA substrates, which differ with respect to the length of the 3′ overhang, indicate that Bax1 cuts specifically 4–6 nt to the dsDNA/ssDNA junction in the ssDNA region. The DNA ladder M was generated by mixing each 10 nm of 5′-labeled ssDNAs of known length, in this case 50, 40, 32, 30, 26, and 19 nt. C, overview of the different substrates used for the incision assays. Arrows depict where Bax1 cuts the DNA substrates.

FIGURE 3.

The endonuclease activity is specific to Bax1. A, Bax1 elution profile after size exclusion chromatography. Absorptions at 260 and 280 nm are shown in gray and black, respectively. The SDS gel below indicates the Bax1 content of the different fractions. Both peaks contain Bax1 and revealed a monomer-dimer equilibrium according to a calibration of the column. B, fractions of the monomer peak were subjected to both SDS-PAGE and incision assays. The incised product was quantified and visualized in a bar chart confirming a direct correlation of the amount of Bax1 and the formation of the DNA fragment.

FIGURE 4.

The presence of XPB inhibits the endonuclease activity of Bax1. Incision assays performed with increasing amounts of the purified XPB-Bax1 complex did not reveal an incision product as observed for Bax1 alone (highlighted by an asterisk). The XPB-Bax1 complex was assumed to contain the two proteins in a 1:1 stoichiometric ratio according to analytical size exclusion chromatography and analytical ultracentrifugation. 10 nm substrate NDT/NDB22 was used throughout.

FIGURE 5.

Identification of functionally important residues. A, conservation of Bax1 among archaea. Multiple sequence alignment of Bax1 (T. acidophilum) and related proteins from Picrophilus torridus, P. furiosus, Pyrococcus abyssi, Haloquadratum walsbyi, S. solfataricus, and Methanosarcina acetivorans depict the level of sequence conservation among different archaea. The arrows point to sites where point mutations were introduced, dots correspond to similar residues, colons correspond to conserved residues, and asterisks correspond to identical residues, respectively. Numbering above the alignment corresponds to the T. acidophilum sequence. B, conserved residues of Bax1 and TspRI endonucleases. The alignment of Bax1 and TspRI endonuclease reveals a high local sequence similarity indicating conserved and thus probably important residues for Bax1 endonuclease activity. C, incision activity of Bax1 mutants. Quantification of three independently conducted incision assays with DNA substrate NDT/NDB26 (2 nm) reveals different cutting efficiencies for diverse Bax1 variants (8 μm each). Error bars represent the S.D. of three independent measurements.

FIGURE 6.

Mg²⁺ is crucial for Bax1 incision activity. Incision assays were performed in the presence of different divalent cations or the chelating reagent EDTA (10 mm each) using the DNA substrate NDT/NDB22 (10 nm). Only in the presence of MgCl₂ is a specific incision product formed.