Poly (ADP-ribose), ADP-ribosylation of proteins and regulation of cell activity]

The nature of a before unknown biological activity of NAD as a substrate in protein modification reaction is considered. Upon enzymatic digestion of NAD its adenosinediphosphate ribose (ADPR) part is transferred to acceptor proteins. ADPR in its mono- or polymeric form is covalently linked to proteins at the expense of NAD's high energy bound. Negatively charged ADPR, in association with a protein, is able to alter the charge, conformation and biological activity of the latter. The reaction is important in structural rearrangements of chromatin, in the synthesis and repair of DNA, in cell growth and differentiation and in the mechanisms of actions of actions of bacterial toxins.     

A structural characterization of in situ chromatin on cell lines isolated from patients affected by ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by numerous clinical and cellular features. The pleiotropic nature of the AT syndrome attests to the multiple roles of ATM, the protein codified by the gene altered in AT patients. We investigated if different mutations of ATM could reflect on different alterations of nuclear architecture and chromatin organization. We selected three lymphoblastoid cell lines isolated from AT patients affected by different mutations of ATM gene and one healthy control. We characterized the in situ chromatin structure of each cell line by a biophysical approach: (1) we evaluated the rearrangements of the chromatin domains at the level of single cell by quantitative fluorescence microscopy; (2) we analysed the changes of the average chromatin condensation by differential scanning calorimetry. The results show that the three different ATM mutations produce significant modifications of both nuclear architecture and chromatin condensation.     

Sequential changes in the macrostructure and RNA-synthesizing activity of nucleolar and extranucleolar chromatin from rat liver cells during induction of DNA synthesis

The dynamics of structural changes and RNA-polymerase activity in rat liver cell chromatin caused by drastic changes in the rates of protein synthesis was investigated. Inhibition of protein synthesis after a single injection of animals with cycloheximide (0.3 mg/100 g of body weight) increased the total condensibility of chromatin. Under these conditions, the stepwise activation of RNA-polymerases I and II correlated with decondensation of chromatin. By the 6-12th hour following cycloheximide injection, a chromatin fraction enriched with RNA-polymerase I and a RNA-polymerase II-rich fraction could be isolated from liver cell nuclei.     

Periodic changes of chromatin organization associated with rearrangement of repair patches accompany DNA excision repair of mammalian cells

We have used 8-methoxypsoralen to probe the chromatin structure of mammalian cells in situ while they repair pyrimidine dimers or bulky lesions in DNA. We observed that excision repair of these DNA lesions is accompanied by periodic alterations of chromatin organization. In parallel, fluctuations of the rates of repair patch synthesis accompanied these structural changes. Taking advantage of the accessibility of free DNA domains for psoralen intercalation, we have developed a technique to quantitatively isolate the micrococcal nuclease-sensitive, free DNA fraction of native bulk chromatin. We have determined the location of newly synthesized repair patches relative to free DNA domains as a function of repair time. Extensive rearrangements of repair patches from these domains into micrococcal nuclease-resistant DNA were observed. Our results indicate that periodic changes of chromatin organization associated with rearrangement of repair patches accompany the process of excision repair in mammalian cells.     

Sphingomyelin synthase in rat liver nuclear membrane and chromatin.

The presence of phospholipids in chromatin has been demonstrated, as well as the difference in composition and turnover compared to those present in the nuclear membrane. Recently, some enzymes were also evidenced in chromatin: the base exchange protein complex and neutral sphingomyelinase. The latter has a particular relevance, since sphingomyelin is one of the phospholipids more represented in chromatin. We therefore decided to study the synthesis of sphingomyelin in chromatin and in nuclear membrane isolated from liver nuclei. The evaluation of the enzyme was made (i) using [(3)H]phosphatidylcholine as donor of radioactive phosphorylcholine and (ii) by identifying the product isolated by thin layer chromatography. In both fractions the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase was present, although with higher activity in nuclear membrane. The enzyme present in the chromatin differs in pH optimum and K(m), showing a higher affinity for the substrates than that of nuclear membrane. The results presented show that sphingomyelin synthase is present not only in the cytoplasm at the level of the Golgi apparatus, but also in the nuclei, at the level of either the nuclear membrane or the chromatin.     

Effects of heat shock on gene expression and subcellular protein distribution in Chinese hamster ovary cells.

Incubation of Chinese Hamster Ovary (CHO) cells for one hour at 43 degrees C results in several obvious changes in protein distribution and protein synthesis. One major protein of the cytoplasm (molecular weight 45,000 daltions), also present as a minor component in the nucleus, rapidly disappeared while several proteins, especially high molecular weight peptides, were induced by heat shock. Localization of the proteins in the cytoplasm, extra-nucleolar chromatin and nucleolar bodies has been carried out. Different sets of induced proteins appear in each subcellular compartment. Four hours after restoration of the normal temperature, the normal pattern of protein synthesis was observed. The 45,000 dalton protein reappeared first. Relations between structural and functional alterations and changes in protein distribution are suggested.     

Modular paths to 'decoding' and 'wiping' histone lysine methylation

Specific cell activity results from developmental and environmental control over the expression of our genes. A key component in epigenetic forms of biological regulation is the methylation of lysine residues in histone proteins. This post-translational modification of chromatin has been vigorously studied over the past few years. Highly specific enzymes catalyzing the synthesis and targeted removal of methyl marks, as well as protein motifs recognizing distinct methylated lysines, have been identified. Here, we provide a molecular overview of discrete structural mechanisms that allow these modular proteins to effect and recognize particular lysine methylation imprints on the chromatin polymer.     

Relationship between chromatin structure and replication in mouse L-cells

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.     

Turnover of chromatin proteins in rat liver during induction of RNA synthesis by electrostimulation of the hypothalamus

It has been shown that the induction of D-RNA synthesis in rat liver nuclei by electrostimulation of hypothalamus is accompanied by a decrease in chromatin protein synthesis and an increase in phosphorylation and acetylation of chromatin proteins. The decrease of the histone synthesis is mainly due to the decrease of [14C]lysine and [14C]alanine incorporation into histones H1 and H4. The relationship between H1, H2b-H3, H2a and H4 histone fractions remains unchanged. Electrostimulation of hypothalamus increases acetylation of H2a and H4 histone fractions and phosphorylation of all histones with the exception of histone H1.     

Genomic variability of sugar beet morphogenic and non-morphogenic callus

Cytogenetic characters and the level of DNA methylation were studied in sugarbeet callus cultures with different morphogenic potential which were obtained from the same leaf explant. It was shown that non-morphogenic callus in comparison with the morphogenic one was characterized by the higher level of cell ploidy and of the frequency of chromosome structural rearrangements even at the early stages of cultivation. Overspiralization of chromosomes and formation of condensed chromatin were observed in polyploid cells of non-morphogenic callus. At the same time cytosine hypermethylation occurs in site-specific CpCpGpG-sequences of DNA indicating the possible role of this DNA modification in the processes of genome rearrangements.     

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.     

Vitellogenin-specific Non-histone Chromatin Protein

A non-histone chromatin protein with a molecular weight of about 10,000 daltons was purified from the liver of egg-laying hens. The protein binds tightly to hen genomic DNA fragments carrying at least a part of the vitellogenin structural gene. This vitellogenin-specific non-histone chromatin protein is induced by estrogen before, or in parallel with vitellogenin synthesis in the liver of male chickens, in which vitellogenin is not usually synthesized.     

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.     

Effect of DNA groove binder distamycin A upon chromatin structure

Background

Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat.

Methodology/Principal Findings

Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods - dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin.

Conclusions/Significance

We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.