Browsing by Author "Haber, Michelle"

The proportion of sheared rat liver DNA recovered from benzoylated DEAE-cellulose in the final stage following stepwise elution with NaCl and caffeine solutions was dependent upon the DNA isolation procedure. An increase in the proportion of DNA containing single stranded regions, consequent upon delay or addition of Mg2+ prior to phenol extraction, suggested nuclease mediated degradation. Administration of methyl methanesulphonate to rats resulted in a consistent proportional increase in the caffeine-eluted fraction. The results of caffeine gradient elution of control and alkylated DNA from benzoylated DEAE-cellulose were consistent with repair-associated single stranded regions being substrates for endogenous single strand-specific exonucleases.

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.