DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.

Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.     

Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions

We report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone gamma-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. However, oxygen did not inhibit formation of the thymine-tyrosine cross-link in gamma-irradiated chromatin or in chromatin treated with H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ ions. By contrast, H2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ ions. Almost complete inhibition of cross-link formation was provided by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide when H2O2/chelated metal ions were used. On the other hand, scavengers only partially inhibited formation of cross-links when H2O2/unchelated metal ions were used, possibly indicating the site-specific nature of cross-linking. Superoxide dismutase afforded partial inhibition only when chelated ions were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a hydroxyl radical induced DNA-protein cross-link involving thymine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving thymine and tyrosine in nucleohistone is described. Hydroxyl radicals were generated in N2O-saturated aqueous solution by ionizing radiation. Samples of nucleohistone were hydrolyzed with HCl and trimethylsilylated. Analysis of irradiated samples by gas chromatography-mass spectrometry with selected-ion monitoring showed the presence of a thymine-tyrosine cross-link on the basis of typical fragment ions from the previously known mass spectrum of its trimethylsilyl derivative. The yield of this DNA-protein cross-link in nucleohistone was measured at incrementing doses of radiation and found to be a linear function of radiation dose between 14 and 300 Gy (J.kg-1). This yield amounted to 0.003 mumol.J-1. The mechanism of formation of this DNA-protein cross-link is thought to result from H atom abstraction by hydroxyl radicals from the methyl group of thymine followed by the addition of the resultant thymine radical to the carbon 3 position of the tyrosine ring and subsequent oxidation of the adduct radical.     

Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

Repair of DNA-protein cross-links in mammalian cells

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).     

cis-diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle

The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.     

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A gamma-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J.kg-1). This yield amounted to 0.05 nmol.J-1. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.     

The action of benzimidazole derivative preparations on the formation of DNA-protein cross-links in the UV irradiation of chromatin

Effect of benzimidazole-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazole interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1.     

Molecular mechanisms of the radiation destruction and formation of cross links in chromatin in aqueous solutions

The modified gel-electrophoresis techniques were used to study DNA destruction in oligonucleosomes of the chromatin and the formation of DNA-protein cross-links under the effect of 60Co-gamma-rays. The yields of DNA destruction were evaluated in different conditions of chromatin irradiation: they were comparable with the yields of single-strand breaks. The bonds in the DNA-protein polymer formed were found to be covalent. It was shown that the processes of formation of cross-links and peroxide radicals (hydroperoxides) were mutually exclusive.     

Initiation of repair of DNA-polypeptide cross-links by the UvrABC nuclease

Although the biochemical pathways that repair DNA-protein cross-links have not been clearly elucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptides constitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test the validity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links were engineered via linkage at (1) an acrolein-derived gamma-hydroxypropanodeoxyguanosine adduct and (2) an apurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteins UvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16 kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined, DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiester bond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater than that measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates were dependent on both the site of covalent attachment on the DNA and the size of the bound peptide. Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slower than the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicating that (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptide cross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggest that proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitating NER.     

Chromatin compaction and the efficiency of formation of DNA-protein crosslinks in gamma-irradiated mammalian cells.

Chromatin has been prepared from Chinese hamster V79 cell nuclei by successive suspension and sedimentation in buffers of decreasing ionic strength. For buffer concentrations from 50 to 1 mM, the resultant chromatin maintained a normal histone content, nucleosomal organization, and attachment to the nuclear matrix; however, as the buffer concentration was reduced from 50 to 10 and 1 mM, the higher-order chromatin structures became increasingly relaxed. Fully expanded chromatin is 5- to 10-fold more susceptible to the induction of DNA-protein crosslinks (DPCs) by gamma radiation than is chromatin residing in living interphase cells. As much as 60-70% of expanded chromatin can be induced to form DPCs as compared to a maximum of about 20% of cellular DNA. For expanded chromatin, the maximum level of induced DPCs is two to three times higher than would be expected if only matrix-associated DNA were induced to form DPCs. Therefore, DNA in distal regions of chromatin loops must also be induced to form DPCs with histones or other nonhistone chromosomal proteins. The hypersensitivity of isolated chromatin to radiation-induced production of DPCs appears to be related to the expansion of chromatin conformation rather than to the removal of intracellular radical scavengers for the following reasons: (a) there is an inverse relationship between the buffer concentration in which the chromatin is suspended and DPC formation, and (b) the induction of a more compact 30-nm chromatin fiber from the expanded 10-nm chromatin fiber in the presence of a low concentration of MgCl2 results in a marked reduction in DPC formation. The formation of radiation-induced DPC seems to occur at maximum efficiency in fully expanded chromatin, since DPC formation cannot be further stimulated by the addition of Cu2+, which can catalyze the production of OH by Fenton chemistry. It is concluded that radiation-induced DNA damage production is greatly influenced by chromatin conformation, and that chromatin as it exists in the cell is a relatively poor substrate for DNA-protein crosslinking in comparison to completely expanded chromatin.     

Stability of DNA-protein interactions in chromatin fractions with different sensitivity to nucleases

It was revealed by means of nucleoprotein-celite-chromatography that DNA-protein interactions in the chromatin fraction sensitive to micrococcal nuclease and DNase II are weaker that in the resistant one. The micrococcal nuclease destroys the DNA-matrix bond resistant to salt-urea, while DNase II does not change the DNA-matrix integrity. Tightness of the DNA-protein interactions is weakened by the increasing chromatin fragmentation, but does not depend on the size of chromatin particles.     

DNA- and RNA-binding proteins of chromatin from Escherichia coli

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sj?stad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.     

运用染色质免疫沉淀技术筛选出AP-2α的靶基因GALK1

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.与其他方法相比,染色质免疫沉淀技术 (chromatin immunoprecipitation assay, ChIP ) 是一种近来研究体内 DNA 与蛋白质相互作用的最好方法之一.本研究利用 ChIP 克隆方法, 找出了 AP-2α所调控的新的下游靶基因 GALK1,并应用 Luciferase assay和 RT-PCR 实验进行了初步的验证.这一新发现,有利于我们进一步研究转录因子AP-2α的功能.     

UV-induced formation of DNA-protein cross-links in chromatin in the presence of chemical agents

Hindered phenols, quinones, and SH-compounds have been studied as possible protectors and sensitizers of DNA-protein cross-linking in chromatin. Efficacy of cross-linking was estimated by the quantity of protein linked to DNA after UV irradiation at 254 um in the presence of chemical agents. Phenols and quinones exert protective influence on cross-linking whereas DNA-protein cross-links are sensibilized by cysteine hydrochloride.     

The use of ultraviolet light in the fractionation of chromatin containing unsubstituted and bromodeoxyuridine-substituted DNA.

Two procedures are described for the fractionation of chromatin containing unsubstituted (LL) DNA and DNA unifilarly substituted with bromodeoxyuridine (HL). The two procedures rely upon the sensitivity of bromodeoxyuridine-containing DNA to UV light to induce either strand breakage or protein crosslinking. When a mixture of LL and HL chromatin is irradiated with UV light, the HL DNA fragments into molecules of smaller molecular weight than the LL DNA and crosslinks more chromosomal protein than the LL DNA. LL and HL chromatin can be fractionated on the basis of size by centrifuging through a neutral sucrose gradient. The HL DNA-protein adducts that are generated by the UV light have a unique buoyant density and may be isolated by isopycnic centrifugation in CS2SO4. The ability to fractionate LL and HL chromatin permits certain studies on the structure of replicating chromatin.     

Evidence that O6-alkylguanine-DNA alkyltransferase becomes covalently bound to DNA containing 1,3-bis(2-chloroethyl)-1-nitrosourea-induced precursors of interstrand cross-links

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA resulted in formation of a DNA-protein covalent complex. Complex formation required active alkyltransferase and brief treatment of DNA with the drug. DNA lost its capacity to form the complex once drug-induced DNA interstrand cross-links were completely formed. These results are consistent with a model in which the transferase catalyzes cleavage at O6-guanine and transfer of the alkyl moiety in a putative O6, N1-ethanoguanine intermediate of cross-link formation. DNA-protein complex formation presumably results when the transferase accepts the N1-ethanoguanine-DNA structure, analogous to its acceptance of simple alkyl groups.     

Preferential formation of UV-induced DNA-protein crosslinks in the nuclear matrix of Ehrlich ascites carcinoma cells

It was shown that after UV-irradiation of Ehrlich ascites tumor cells with doses suppressing DNA replication, DNA-protein cross-links were mainly predominantly in the nuclear matrix as compared to peripheral chromatin. A modified method of determining DNA-protein cross-links in the nuclear matrix preparations is proposed.