Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.
HIGHLIGHTS-APE1 forms a stable complex with ssDNA longer than 15 mer; -APE1-catalyzed repair and TF-stimulating activities depend on the structure of DNA duplex; -APE1 acts synergistically with reducing agents to stimulate DNA binding of AP-1 and p50; -APE1 stimulates DNA binding of p50-C62S redox-insensitive mutant; -APE1 forms stable multiprotein oligomers on intrinsically curved DNA regions.
ABBREVIATIONSROS, reactive oxygen species; AP, apurinic/apyrimidinic site; THF, 3-hydroxy-2hydroxymethyltetrahydrofuran or tetrahydrofuran; αdA, α-anomeric 2′-deoxyadenosine; Z, 3nitropyrrole; BER, base excision repair; NIR, nucleotide incision repair; APE1 (a.k.a. APEX1, HAP-1, or Ref-1), human major AP endonuclease 1; Nfo, Escherichia coli endonuclease IV; ARP, Arabidopsis thaliana major AP endonuclease; wARP, wheat Triticum aestivum AP endonuclease. Milena BAZLEKOWA-KARABAN et al., Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1.
Xylanase is one of industrial enzymes with diverse applications including the paper-bleaching industry and feed additives. Here, a strain having xylanolytic activity and identified as Bacillus sonorensis T6 was isolated from soil. A secretory enzyme was identified by mass-spectrometry as a xylanase of glycosyl hydrolase family 11, with a molecular weight of 23.3 kDa. The xylanase gene of Bacillus sonorensis T6 was cloned and expressed in Escherichia coli (yielding an enzyme designated as rXynT6-E) and in Pichia pastoris (yielding rXynT6-P). The recombinant xylanases were found to have optimal activity at 47–55°C and pH 6.0–7.0. The recombinant xylanase expressed in P. pastoris has 40% higher thermal stability than that expressed in E. coli. The recombinant xylanases retained 100% of activity after 10 h incubation in the pH range 3–11 and 68% of activity after 1 h at pH 2.0. The xylanase activities of rXynT6-E and rXynT6-P under optimal conditions were 1030.2 and 873.8 U/mg, respectively. The good stability in a wide range of pH and moderate temperatures may make the xylanase from Bacillus sonorensis T6 useful for various biotechnological applications, e.g., as an enzyme additive in the feed industry.
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