Activation and Enzyme Characteristics of a DNA‐Restrained Phosphatase in Chromatin‐Associated Complexes

Abstract

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 adeno$inetriphosphatase/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 nontrivial 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 Mgz', 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.Keywords: adenosinetriphosphatase ; phosphatase ; non-histone proteins ; chromatin structure ; chromosome territories.Recent chromosome-painting results based on in situ hybridization with chromosome-specific composite and gene-specific probes confirm that interphase chromosomes and individual genes occupy distinct territories and sites in the interphase cell nucleus [I]. These findings are in agreement with a highly ordered topological organization of the genome, and they support the view that the intranuclear positioning of genes is likely to be involved in epigenetic regulation of gene expression [2, 31. Further support for this hypothesis is expected to come from biochemical characterization and analysis of DNA-polypeptide complexes involved in the three-dimensional genome organization, e.g. in the configuration of the volumes and shapes of the territories occupied by interphase chromosomes. Recently, we detected unusually tight but non-covalent DNA-polypeptide complexes that are supposed to meet these criteria [4]: DNA isolated by a method avoiding phenol extraction [5] resolves efficiently deproteinized DNA. However, this DNA remains associated with globular non-histone protein complexes that consist of three major polypeptides (62, 52, and 40 kDa) which are not released from DNA during further deproteinization procedures including repeated salting out steps, prolonged incubation of DNA in 1 % ...

Tightly bound to DNA proteins: Possible universal substrates for intranuclear processes

“…Later the additional minor TBPs of 62, 52 and 40 kDa were described and it was reported that these proteins assemble in globules 12.8 nm in diameter (Loeffler et al, 1996). Similar globular structures were described for TBPs localized in interaction sites with the nuclear matrix, besides DNA and proteins (7 or 8 polypeptides) the globules contained RNA molecules (Chernokhvostov and Georgiev, 1990;Chernokhvostov et al, 1986Chernokhvostov et al, , 1989.…”

“…Active enzyme was formed by subunits of 56 and 59 kDa (Loeffler et al, 1996;Juodka et al, 1995b). Later protein kinase activity was also found in the TBPs fraction of these cells.…”

A multifunctional protein: involvement of the alpha‐1 serum protease inhibitor in SDS and high salt‐stable DNA‐protein complexes

“…The electrophoretic mobilities, the glycoprotein nature [1, 7], the overlapping 12mer microsequence (Fig. 2) and the immunochemical data (Figs.…”

“…Complexes of this type were found to be attached to DNA isolated from human placenta and from murine tumor cells [6]. The DNA‐protein complexes survive treatments designed to dissociate DNA‐protein complexes, including cell lysis in the presence of SDS and proteinase K, salting out steps, prolonged incubation in 1% SDS or 4 M urea at 56°C and ethanol precipitations [6, 7]. Further characterization of the 52‐kDa nuclear glycoprotein by partial sequencing and by application of immunochemical and recombinant DNA techniques indicates that it is identical with the α‐1 protease inhibitor which points to a nuclear function of this protein.…”

“…However, these results were not verified by other methods and the significance of this potential intracellular location of α‐1 protease inhibitors and their subcellular distributions have not yet been investigated. Studying tight DNA‐protein complexes we characterized a supramolecular structure consisting of three major glycosylated polypeptides with apparent molecular masses of 62 kDa, 52 kDa and 40 kDa [6, 7]. Complexes of this type were found to be attached to DNA isolated from human placenta and from murine tumor cells [6].…”