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Nitric oxide-induced modifications of DNA occur either by directly altering DNA chemically through reactive nitrogen oxide species (RNOS) or indirectly by inhibiting various repair processes. DNA ligases are enzymes which rejoin single-strand breaks and are critical for DNA integrity during processes such as gene transcription and repair. The eukaryotic and T4 DNA ligases are active in the presence of ATP and act in two steps: the formation of protein-AMP intermediates, then the ligation of DNA breaks. When T4 DNA ligase was exposed to the NO generator DEA/NO (Et2N[NO(NO)]Na), a concentration- and time-dependent inhibition of these two steps, adenylylation of the protein and ligation of the substrate, was observed. This inhibition was abated by the presence of cysteine, suggesting that RNOS, rather than NO, mediated the inhibition of the ligase activity. As mammalian and T4 DNA ligases act by the same mechanism, the inhibition of DNA ligase may explain the increase in single-strand breaks reported for cells exposed to NO and provides a mechanism to increase DNA lesions without direct chemical modification of DNA by NO or RNOS.

The appearance of DNA replication intermediates was investigated in a human fibroblast strain (46 BR) which is hypersensitive to the lethal effects of 3-aminobenzamide. 3-Aminobenzamide is an inhibitor of poly(ADP-ribose) synthetase and modulates DNA ligase activity. We detected the same intermediates (10 kb DNA and Okazaki-fragments) as in normal fibroblasts, but kinetics and amounts of intermediates were altered, either as a result of, or in order to overcome the defect in the cells.

A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. In activities of the two largest species, DNA ligase Ia (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50 degrees C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases Ia and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.

Following treatment of human fibroblasts with dimethyl-sulphate, more breaks persisted in DNA in cells incubated with 3-aminobenzamide, an inhibitor of ADP-ribosyltransferase, than in its absence. This effect of 3-aminobenzamide was more pronounced in non-dividing than in dividing cells. If non-dividing cells were treated with dimethylsulphate and then incubated for a few hours in the absence of 3-aminobenzamide, few breaks were detectable in the DNA. Subsequent addition of 3-aminobenzamide resulted in the reappearance of many breaks in the DNA. These data suggest that continued synthesis of poly(ADP-ribose) reduces the steady state level of breaks during excision repair of alkylation damage. This is probably mediated by the stimulation of DNA ligase activity. Inhibition of poly(ADP-ribose) synthesis with 3-aminobenzamide maintains or restores a higher steady-state level of breaks.