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-’. 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),. 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 cpX 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. Repair endonucleases which recognize damaged regions in DNA induced by chemicals, ultraviolet light or y-irradiation have been purified from several mammalian sources and the specificities of these enzymes have been studied in vitro [l - 51. Except for the high-molecular-mass and partly unstable enzyme isolated from calf thymus [4], the enzymes require as a substrate a DNA treated with a high dose of ultraviolet light in order to introduce nicks. It is known that high doses of ultraviolet light also introduce non-dimer modifications in the DNA [6]; because of this it has been claimed that these enzymes are not specific for pyrimidine dimers. Moreover, since the enzymes from mouse cells [5] and from human cells [3] also act on damage caused by Os04 treatment of the DNA, it has been suggested that these enzymes recognize thymine glycol residues in DNA. In the case of the enzyme from mouse plasmacytoma cells we have shown that the ultraviolet-light-damaged DNA must be in a supercoiled form in order to act as a substrate for the purified enzyme. The human enzyme studied by Brent [3], as well as the mouse enzyme studied in our laboratory, both have an associated weak AP-endonuclease activity. This differs from the major AP-endonucleases found in these cells since is does not require Mg2+ for its activity. It has not been possible to separate the ultraviolet light endonuclease and the APendonuclease activities. Heat denaturation studies indicate that the two activities are located on the same protein. Brent [3] has postulated that the human endonuclease acta in a way