Properties of a DNA repair endonuclease from mouse plasmacytoma cells

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Abstract
The properties of a DNA repair endonuclease isolated from mouse plasmacytoma cells were studied. It acted on UV-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at UV 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 UV-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 UV-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3''-OH ends. Treatment of the nicked UV-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 labeled 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 molecules; it produced random nicks in UV-light-modified .rho.X174 replicative form I DNA. Antibodies raised against UV-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. The enzyme apparently has a specificity directed toward helical distortions.