High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

Juodka, Benediktas; Spieß, Eberhard; Angiolillo, Antonella; Joswig, Gaby; Rothbarth, Karsten; Werner, Dieter

doi:10.1093/nar/23.8.1359

Cited by 18 publications

(31 citation statements)

References 17 publications

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“…Although benzonase has been extensively reported to digest both chromatinassociated and nonassociated DNA, we verified the effectiveness of benzonase to digest chromatin-associated nucleic acid, which is required for complete removal of viral DNA not protected by a capsid ( Fig. 2A and B) (1,24,29,49). Aliquots of 20-day CVPs were digested with benzonase for up to 3 h. The viral and cellular DNA was then extracted, followed by SYBR green-based qPCR.…”

Section: Resultsmentioning

confidence: 74%

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Conway¹,

Alam²,

Ryndock³

et al. 2009

J Virol

189

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Papillomavirus capsids are composed of 72 pentamers reinforced through inter-and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14,15,30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14,15,30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ϳ8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a Tϭ7 icosahedral lattice (2,9,17,19,36,42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6,12,25,40,43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7,20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation...

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Section: Resultsmentioning

confidence: 74%

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Conway¹,

Alam²,

Ryndock³

et al. 2009

J Virol

189

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“…DNA isolated by the salting out procedure [5] is essentially deproteinized. However, it remains associated with polypeptide complexes composed of three major polypeptides (40, 52, and 62 kDa) [4]. Following nuclease (deoxyribonuclease 1 or benzonase) digestion of 10-20 A,,,, units (1 mg) of DNA, these polypeptides can be visualized on SDS/polyacrylamide gels by Coomassie brilliant blue staining (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The isolation of DNA by the salting out procedure and optional modifications of this procedure have been described previously in detail [4]. Here we used the following steps which are appropriate to characterize the encrypted phosphatase.…”

Section: Methodsmentioning

confidence: 99%

“…3A-C). Since the R, values of AMP and inorganic phosphate are rather similar, the ATP-degrading enzymatic activity could be suggested to reflect an adenosinetriphosphatase activity [4]. However, at higher substrate concentrations and shorter reaction times a clear phosphate-specific peak was resolved which indicated that the polypeptides which were isolated with the DNA and released from the DNA by deoxyribonuclease I digestion comprise an enzyme with phosphatase characteristics (Fig.…”

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confidence: 99%

“…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 % SDS or 4 M urea at 56"C, and ethanol precipitations.…”

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

Loeffler

Spieß

Juodka

et al. 1996

European Journal of Biochemistry

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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 % ...

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Modified salting-out method for DNA isolation from newborn cord blood nucleated cells

Noguera

Tallano

Bragós

et al. 2000

J. Clin. Lab. Anal.

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The present work describes modification of a widely used salting‐out procedure to rapidly extract DNA suitable for PCR, using the ARMS method to amplify a target sequence in the β‐globin gene. The salting‐out DNA extraction procedure did not completely remove or decrease the presence of inhibitors to PCR in a considerable number of cord blood samples. By introducing a simple phenol/chloroform step, before ethanol precipitation of the nucleic acid, to certain samples, we were able to eliminate or substantially reduce the presence of inhibitors to PCR without having to re‐extract the samples. J. Clin. Lab. Anal. 14:280–283, 2000. © 2000 Wiley‐Liss, Inc.

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High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

Abstract: Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller,S.A., Dykes,D.D. and

Cited by 18 publications

References 17 publications

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

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

Modified salting-out method for DNA isolation from newborn cord blood nucleated cells

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