Expression in Escherichia coli of a rat cDNA encoding an apurinic/apyrimidinic endonuclease.
A rat cDNA (rAPEN) with 85% DNA identity to the major human apurinic/apyrimidinic (AP) endonuclease gene was used to construct a fusion between it and glutathione-S-transferase (GST). The GST-rAPEN fusion was subsequently overexpressed in Escherichia coli, purified on glutathione-agarose affinity columns, and the purified protein tested for AP endonuclease activity. DNA nicks were found to be specifically introduced into AP DNA in a reaction that was dependent upon the time of incubation and the amount of GST-rAPEN added. The DNA scissions produced by GST-rAPEN were determined to be adjacent and 5' to an AP site. The purified fusion protein was also able to efficiently remove 3'-(4 hydroxy-5-phospho-2-pentenal) residues, and to a lesser extent 3'-phosphoglycolate residues. The GST-rAPEN activity failed to exhibit any 3'-5' exonuclease activity, a characteristic shared by the major AP endonuclease in bovine and human.
D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D018076:DNA, Complementary / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase