The effect of preincubation of HeLa cell nuclei with ATP on the degradation of mononucleosomal DNA by micrococcal nuclease

Abstract

HeLa cell nuclei with DNA labeled with [ H] thymidine have been preincubated under varying conditions and then incubated with micrococcal nuclease. Aliquots, removed at increasing times, were analyzed for mononucleosomal size DNA and for acid-soluble DNA, the ratios were plotted and a slope was determined. Preincubation with ATP and a regenerating system increased the slope 2 fold. Optimum ATP concentrations were above 0.25 mM. The ATP effect was reversed by novobiocin. No inhibition of the ATP effect was observed with nalidixic acid, coumermycin, oxolinic acid, VM-26, aphidicolin, or 3 aminobenzamide. NAD or cAMP or cGMP had no effect with or without ATP. Other nucleoside triphosphates could substitute to varying degrees for ATP as could ATP analogues. Nuclei from log phase cells showed no ATP effect, but log phase cells, partially depleted of ATP by incubation with deoxyglucose, showed the effect. The effect was lost in nuclei on long-term storage. No evidence was found for differential degradation of core histones, histone H-1 or DNA, and there was no evidence of nucleosome sliding.