Activation and enzyme characteristics of a DNA-restrained phosphatase in chromatin-associated complexes

DNA-bound polypeptide complexes composed of several non-histone polypeptides that resisted harsh DNA deproteinization procedures were characterized. The three major polypeptides of these complexes have molecular masses of 62, 52, and 40 kDa. They constitute supramolecular structures that reside on isolated DNA in dense clusters. The supramolecular complexes were released from DNA as globular 12.8 +/- 0.8-nm particles; these particles were gradually disassembled to form smaller supramolecular structures. The DNA-bound complexes comprise of an encrypted adenosinetriphosphatase/phosphatase activity, which is a minor but intrinsic component of the complexes. The enzyme remained inactive as long as the complexes were bound to DNA. However, the enzyme was activated concomitantly with the progression of DNA digestion, which indicated that DNA was involved in the downregulation of the enzyme. The inactive DNA-restrained complex could not be restored in vitro, which indicated its non-trivial nature. Once released from DNA, the enzyme was inactivated over a period of several hours. However, in the DNA-associated complexes its potential to become activated during DNA digestion was conserved for several months. In the activated state, the enzyme showed an optimum activity at pH 9.5, was stimulated by Mg2+, inhibited by vanadate and EDTA, but was not significantly inhibited by okadaic acid. The active enzyme, which consists of two subunits of 56 kDa and 59 kDa, can be released from the supramolecular structures by agarose gel electrophoresis. A regulatory mechanism therefore exists for the downregulation of this phosphatase by DNA.