We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.
Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)
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