Takanori Tomura

Purification and some properties of a mammalian repair endonuclease

Abstract An endonuclease has been purified from rat liver to electrophoretic homogeneity on polyacrylamide gel. The elution pattern of the purified enzyme on the G75 Sephadex column suggests a molecular weight between 15000 and 20000. Sedimentation of substrate on neutral and alkaline sucrose gradients after incubation with the purified enzyme, or incubation of the 3 2P-labeled substrate with purified endonuclease and alkaline phosphatase demonstrated that the enzyme causes single strand nicks to appear in ultra violet- and acetylaminofluorene-bound double stranded DNA. The purified preparation is devoid of exonuclease and alkaline phosphatase activity and is without effect on denatured DNA. The nicked substrate becomes susceptible to an attack by bacterial DNA polymerase I as shown by a release of material absorbing at 260 nm, in the acid soluble fraction and an increased priming ability of the substrate for DNA polymerase I. The sequential attack of the substrate with the purified endonuclease, alkaline phosphatase and DNA polymerase I releases thymine dimers in the case of ultra violet irradiation and acetylaminofluorene base complexes in the case of acetylaminofluorene-bound DNA in the acid soluble.

Mutagenicity of reactive derivatives of carcinogenic hydrocarbons: Evidence of DNA repair

Abstract The ability of cellular DNA repair enzymes, which are active on ultraviolet light-induced lesions in DNA, to recognize and repair damage induced in DNA by exposure to carcinogenic polycylic hydrocarbons was investigated and the effect of such repair processes on the mutagenicity of the hydrocarbons determined. The carcinogenic hydrocarbos, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA), chosen for this study because they form well characterized, stable products with DNA, were dissolved at various concentrations in acetone, added under mild conditions to biologically active DNA isolated from Bacillus subtilis , and the reaction stopped by ethanol precipitation. The hydrocarbons were determined by specific radioactivity to be covalently linked to DNA at a frequency of from 1–5 per 1000 nucleotides. An increased frequency of bound hydrocarbon molecules was directly correlated with a decrease in the buoyant density of the DNA as measured in analytical CsCl centrifugation studies. The samples of hydrocarbon-bound DNA were tested for survival of biological activity and for the frequency of induced forward mutations in two recipient strains (hcr + and hcr − ) of Bacillus subtilis which differ in their ability to repair ultraviolet light-induced lesions in DNA. The survival of the biological activity was significantly higher in the repairing strain (hcr + ). A higher frequency of mutations was detected in the repairing strain as well. The loss of transforming activity and the increase in the frequency of mutations (up to 20-fold) was directly proportional to the amount of hydrocarbon bound to the DNA samples. The majority of these mutations proved unable to revert spontaneously. Finally, the ability of highly purified rat liver endonuclease, shown to recognize lesions in UV-irradiated DNA, to recognize such hydrocarbon lesions was investigated. Tritiated 7-BrMeBA-treated DNAs exposed to the enzyme were found to sustain single-strand nicks in proportion to the amount of hydrocarbon bound while untreated DNA remained substantially intact. The action of the endonuclease appeared to result in an increase in the biological activity of DNA containing hydrocarbon residues when this was assayed in the hcr − mutant.

The effect of a mammalian repair endonuclease on X-irradiated DNA

DNA was extracted from rat liver of non-irradiated animals, and was irradiated in vitro, and from animals which received whole body doses of X-radiation. Sedimentation on neutral and alkaline sucrose gradients as well as measurements of 32P release after sequential treatment with endonuclease and alkaline phosphatase and determination of triphosphate incorporation after the sequential treatment with endonuclease, alkaline phosphatase and DNA polymerase indicated that DNA irradiated in vivo and in vitro were effective substrates for the mammalian repair endonuclease. The experiments suggest that in addition to strand breaks, X-radiation causes base damage and they have provided a plausible explanation for the formation of double strand breaks in DNA irradiated in vivo.

The action of a mammalian endonuclease on psoralen-bound DNA

The sequential actions of two enzymes believed to be involved in DNA repair, namely a mammalian endonuclease and the bacterial DNA polymerase I on psoralen bound 32P-labeled DNA, was studied. When ultraviolet-irradiated DNA is exposed to the sequential action of the endonuclease, the formation of single-strand breaks prepares the DNA for the exonucleolytic excision of thymine dimers. The mammalian endonuclease purified from rat liver to electrophoretic homogeneity is inactive on normal DNA, DNA irradiated at 360 nm or DNA mixed with psoralen without irradiation. Incubation of psoralen-bound DNA labeled with 32P with the endonuclease releases the isotope in the acid soluble indicating that psoralen-bound DNA is susceptible to the endonucleolytic attack. Sedimentation of DNA on sucrose gradients indicates that there is no collapse of the DNA molecule after treatment with the endonuclease. Moreover, there is no release of the adduct in the acid soluble after treatment with DNA polymerase, indicating that the 5--3 min exonucleolytic activity of that enzyme is impaired by the remaining crosslinks. The crosslinks also inhibit the incorporation of [3H] dATP in presence of DNA polymerase I.

Purification of human and monkey endonucleases acting on ultraviolet-irradiated DNA

Abstract A human endonuclease was purified from human spleen. The enzyme causes singlestrand breaks in uv-irradiated but not in nonirradiated DNA, and has properties similar to an endonuclease purified from rat liver. A similar enzyme was also partially purified from monkey liver.

Alterations of liver DNA after x-irradiation. Reassociation and hybridization with nuclear RNA

When RNA is extracted either from irradiated or unirradiated rat livers, no difference in its ability to hybridize with DNA is detectable. In contrast, the ability of DNA extracted from irradiated animals to hybirdize with RNA is decreased, probably as a result of DNA fragmentation. Sedimentation of X-irradiated DNA on sucrose gradients separates a small (low molecular weight) from a large peak (high molecular weight). The hybridization capacity for RNA of the large molecular weight DNA is similar to that of unirradiated DNA, but that of the small molecular weight is DNA reduced. After irradiation of the denatured DNA in vitro, the reassociation of DNA is inhibited, which suggests that DNA strands have lost part of their complementariness.