Properties of a DNA repair endonuclease from mouse plasmacytoma cells.
The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.
Helland
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Raae
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Fadnes
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Kleppe
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D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000378:metabolism / Q000528:radiation effects; D004278:DNA, Superhelical / Q000302:isolation & purification; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000528:radiation effects; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000528:radiation effects; D051379:Mice; D010766:Phosphorylation; D010954:Plasmacytoma / Q000201:enzymology*; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D004278:DNA, Superhelical; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase