Immunodetection of DNA-protein crosslinks by slot blotting

Ultraviolet light, formaldehyde, cis-diamminedichloroplatinum(II), chromate (Cr6+), or chromium chloride (Cr3+) under the appropriate conditions caused the formation of DNA-protein crosslinks in intact Chinese hamster ovary (CHO) cells or in cell nuclei. The DNA-protein crosslinks were isolated, applied to nitrocellulose filters, and reacted with antibodies to nuclear proteins. An antiserum to a 97-kD nuclear protein detected p97-DNA complexes in CHO nuclei and cell cultures treated with UV light, cis-Pt and formaldehyde. Exposure to Cr3+ induced p97-DNA crosslinks only in isolated nuclei, while chromate (Cr6+) treatment resulted in significant crosslink formation only in intact cells. Analysis of western blots with the p97 antiserum indicated that crosslinks induced by formaldehyde or ultraviolet light required DNAase I digestion of DNA for migration of the p97 complexes into the gel. In contrast, the 97-kD antigen from the metal-induced crosslinks was released from DNA and resolved in the gel when 2-mercaptoethanol was included in the electrophoresis sample buffer. Assay of slot blots with an antihistone monoclonal antibody indicated that formaldehyde, but not cis-Pt or chromate, crosslinked histones to the DNA. These results illustrate the utility of immuno-slot blots in detecting and characterizing DNA-protein complexes induced by diverse chemical and physical agents.     

A blotting method for monitoring the formation of chemically induced DNA-protein complexes

The formation and identification of DNA-protein crosslinks are usually detected by filter binding assays such as alkaline elution. We describe a modified blotting method to selectively identify DNA-protein complexes (DPCs) formed in vitro by either Cr3+ ion or formaldehyde. This protocol allows DPC formation in vitro to be assayed with various chemical agents, requires minimal usage of radioactivity, and is performed in a shorter time frame than that commonly used to resolve DPCs from free proteins and unbound DNA.     

Chromium-induced cross-linking of nuclear proteins and DNA

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.     

DNA-binding proteins of mammalian mitochondria

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.     

Analysis of EDTA-chelatable proteins from DNA-protein crosslinks induced by a carcinogenic chromium(VI) in cultured intact human cells

DNA-protein crosslinks (DPCs) were induced in intact human leukemic T-lymphocyte MOLT4 cells or isolated nuclei by treatment with potassium chromate, chromium(III) chloride hexahydrate or x-rays. The proteins complexed to DNA were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). A group of identical non-histone proteins was crosslinked to DNA by any of the three treatments, except that a 51 kDa basic protein was additionally complexed to DNA when either potassium chromate or chromium(III) chloride hexahydrate was the crosslinking agent. Treatment of chromate-induced DNA-protein crosslinks with EDTA or thiourea followed by ultracentifugation dissociated the major proteins from the complex indicating that these proteins were crosslinked to DNA by direct participation of a EDTA-chelatable form of chromium such as Cr(III) through sulfur containing amino acid residues. The 51 kDa protein was not seen in the post-EDTA pellet but was present in the post-thiourea pellet, indicating that it was also crosslinked to DNA by Cr(III) through non-sulfur-containing amino acids. Digestion of x-rays-induced DPCs by DNase I also revealed this protein on two-dimensional gels indicating that the same protein was also crosslinked by oxidative mechanisms. The involvement of oxidative mechanisms in the crosslinking process was indicated as the majority of the proteins in chromate-induced DPCs were resistant to EDTA and thiourea treatment, and were found to crosslink to DNA when x-rays were used as the crosslinking agent. These results suggest that the chromate-induced DPCs are formed by the generation of reactive oxygen species during the intracellular chromate reduction as well as by the biologically generated Cr(III). About 19% of DNA-protein crosslinks actually involve Cr(III) crosslinking DNA to proteins, about 14% involve Cr(III) crosslinking DNA to proteins through non-sulfhydryl containing moieties and about 5% involve Cr(III) crosslinking DNA to sulfhydryl groups on proteins. The remaining 81% of DNA-protein crosslinks appear to be oxidatively crosslinked out of which about 45% appear to be through sulfhydryl groups and another 36% appear to be through non-sulfhydryl groups.     

DNA-protein crosslinking by heavy metals in Novikoff hepatoma

Crosslinking of proteins to DNA was studied in live intact Novikoff ascites hepatoma cells exposed in vitro to salts of chromium VI, III, and II, nickel II, cadmium II, and to CoCl2, As2O3, and AlK(SO4)2. DNA-protein complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by polyacrylamide gel electrophoresis. Hexavalent chromium compounds formed DNA-protein complexes very efficiently. The trivalent, poorly soluble, cupric chromite was nearly as efficient crosslinker as hexavalent Cr, perhaps because phagocytosis facilitated its entry into the cells. The more basic divalent form produced hardly any crosslinks. Most of the crosslinked proteins were common to all of the chromium salts employed. Nickel salts formed DNA-protein crosslinks less efficiently. Most proteins crosslinked by this metal had a high molecular weight ranging from 94,000 to 200,000. There was little qualitative difference between the crosslinked protein patterns for several various nickel (II) salts. Similar results were obtained for cells incubated with cadmium salts. Most of the proteins crosslinked by cadmium had high molecular weights and were similar to those crosslinked by nickel (II). Relatively weak, but significant, crosslinking was also observed when the Novikoff hepatoma cells were exposed to CoCl2, As2O3, or AlK(SO4)2.     

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.     

Application of fluorescamine to the study of protein-DNA interactions.

The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes.     

Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

Characterization of DNA sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome.

The terminal 500-base-pair alpha sequence of the herpes simplex virus 1 genome contains signals for cleavage (Pac1 and Pac2) of unit-length DNA molecules from concatemers in unique stretches of sequences designated Ub and Uc, respectively, and a cis site for cleavage designated DR1. We report that nuclear extracts from infected cells contain factors which form two DNA-virus-specific protein complexes with components of the a sequence. Purification of the factors forming the V2 complex yielded a protein with an apparent molecular weight of 82,000 binding to DNA in a non-sequence-specific manner. Addition of Mg2+ to the purified protein-DNA probe mixture resulted in exonucleolytic degradation of the DNA. The protein was identified as the virus-specific DNase with monoclonal antibody specific for the viral enzyme. The purification of the proteins forming the V4 complex yielded two proteins with molecular weights of greater than 250,000 and 140,000 corresponding to infected cell protein 1 and to an as yet unidentified protein, respectively. These proteins formed two DNA sequence-common bands with a number of DNA probes and one sequence-specific band with probes containing both Pac2 and DR1 but not with probes containing either site alone or Pac1 and DR1. Since the DNA probe containing Pac2 and DR1 inserted into viral genome or into amplicons induced specific cleavage of the DR1 sequence whereas the nonreactive probes failed to induce the cleavage, the formation of this sequence-specific DNA-protein complex is significant and may reflect a DNA-protein interaction essential for cleavage. The possible role of the proteins identified in this study for the cleavage-packaging of viral DNA into capsids is presented.     

Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 1. Circular dichroism spectroscopy

Mechanisms of interaction of DNA with nonhistone chromosomal protein HMGB1 and linker histone H1 have been studied by means of circular dichroism and absorption spectroscopy. Both proteins are located in the internucleosomal regions of chromatin. It is demonstrated that the properties of DNA-protein complexes depend on the protein content and cannot be considered as a mere summing up of the effects of individual protein components. Interaction of the HMGB1 and H1 proteins is shown with DNA to be cooperative rather than competitive. Lysine-rich histone H1 facilitates the binding of HMGB1 to DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino acid residues in the C-terminal domain of HMGB1. The observed joint action of HMGB1 and H1 stimulates DNA condensation with the formation of anisotropic DNA-protein complexes with typical ψ-type CD spectra. Structural organization of the complexes depends not only on DNA-protein interactions but also on interaction between the HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the mode of interactions between components in the triple DNA-HMGB1-H1 complex. The binding of Mn²⁺ ions weakens DNA-protein interactions and strengthens protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.     

Reconstitution of intercalator-induced DNA scission by an active component from nuclear extracts

Treatment with intercalating agents causes formation of protein-associated DNA breaks in mammalian cells in culture and in the nuclei isolated from these cells. We found that this effect, when induced by the intercalator m-AMSA, required a component which could be dissociated from nuclei by 0.3 M NaCl. The effect was restored by combining the extracted nuclei with the nuclear extract. The active component of the extract eluted in gel filtration at a point corresponding to a molecular weight of 800 000. During its reaction with DNA, DNA-protein links and DNA breaks appeared in approximately equal frequencies. In this respect the reaction stimulated by m-AMSA resembled the reaction of a topoisomerase with DNA. However, intercalator-stimulated formation of protein-associated DNA breaks differed from the activity of the nuclear topoisomerase I in that there was a different optimum salt concentration and a different apparent molecular weight.     

DNA-binding proteins in cells and membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb fractions BI and BII of Neisseria gonorrhoeae contain both linear and circular DNAs. Because little is known about the interactions between DNA and blebs, studies were initiated to identify specific proteins that bind DNA in elaborated membrane blebs. Western immunoblots of whole-cell and bleb proteins from transformation-competent and DNA-uptake-deficient (dud) mutants were probed with single- or double-stranded gonococcal DNA, pBR322, or synthetic DNA oligomers containing intact or altered gonococcal transformation uptake sequences. The specificity and sensitivity of a nonradioactive DNA-binding protein assay was evaluated, and the assay was used to visualize DNA-protein complexes on the blots. The complexes were then characterized by molecular mass, DNA-binding specificity, and expression in bleb fractions. The assay effectively detected blotted DNA-binding proteins. At least 17 gonococcal DNA-binding proteins were identified; unique subsets occurred in BI and BII. Certain DNA-binding proteins had varied affinities for single- and double-stranded DNA, and the intact transformation uptake sequence competitively displaced the altered sequence from a BI protein at 11 kilodaltons (kDa). A dud mutant, strain FA660, lacked DNA-binding activity at the 11-kDa protein in BI. The segregation of DNA-binding proteins within BI and BII correlates with their distinct protein profiles and suggests that these vesicles may play different roles. Although the DNA-binding proteins expressed in BII may influence the nuclease-resistant export of plasmids within BII vesicles, the BI 11-kDa protein may bind transforming DNA.     

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5'' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.     

Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

DNA–protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA–Pt–protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA–protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug.     

Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light.

Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo.