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 % ...
ATR-Seckel syndrome and ataxia-teleangiectasia are autosomal recessive disorders associated with hematologic malignancies. They share the feature of genetic instability and are caused by defects in the DNA damage response: underlying causes are a hypomorphic ATR mutation and inactivating ATM mutations, respectively. A common substrate of ATM and ATR in the DNA damage response is the nuclear checkpoint kinase Chk1, which we have recently shown to localize to interphase centrosomes and thereby negatively regulate entry into mitosis by preventing premature activation of cyclin B-Cdk1 (Nat Cell Biol6: 884–891, 2004). Here, we demonstrate that DNA damage by ultraviolet radiation or hydroxyurea treatment enhances the centrosomal localization of Chk1. In fibroblasts derived from ATR-Seckel or ataxia-teleangiectasia patients, this DNA damage-dependent centrosomal accumulation of Chk1 was rather more pronounced in comparison with control fibroblasts. Accordingly, this cellular reaction is not dependent on ATR or ATM. These results indicate that centrosomal accumulation of Chk1 might contribute a novel regulatory mechanism to the DNA damage checkpoint. Our study might help to further dissect the contributions of ATR and ATM to the DNA damage response, and to better understand their role in the development of hematologic malignancies.
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