Enhancement of radiation-induced DNA-protein crosslinking by N-methylformamide

The effects of the differentiating agent N-methylformamide (NMF) on radiation-induced DNA damage and repair in vitro were investigated using the alkaline elution assay. Two tumor cell lines were examined: Clone A, a human colon adenocarcinoma, and HCA-1, a murine hepatocarcinoma. Both cell lines showed changes suggestive of a better differentiated phenotype when exposed to NMF. Treatment with NMF enhanced the radiation sensitivity of Clone A cells but had no effect on the radiation response of HCA-1 cells. Irradiation of NMF-treated cells, both Clone A and HCA-1, induced the formation of DNA-protein crosslinks (DPCs). The level of DPCs induced increased linearly as a function of increasing gamma-ray dose. The DPCs did not seem to be the result of NMF exposure alone, but rather an NMF-mediated modification of the spectrum of gamma-ray-induced DNA lesions. When the DPCs were removed by proteolytic digestion, no NMF effect was observed on either strand-break formation or repair.     

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.     

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]- -tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.     

Characterization of DNA--protein cross-links induced by oxanine: cellular damage derived from nitric oxide and nitrous acid

Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide, nitrous acid, or N-nitrosoindoles. It was shown by gel electrophoresis that oxanine mediated the formation of DNA-protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2'-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA-protein cross-links induced by oxanine have not been characterized. In this study, we find that the thiol group of the amino acid side chain is reactive toward oxanine, forming a thioester. Two reaction products of oxanine, namely, the thioester and the amide adducts, with the endogenous tripeptide glutathione (GSH) as a model protein were characterized on the basis of their UV, NMR (1H- and 13C-), and mass spectra. Interestingly, the disulfide GSSG also reacts with oxanine, forming the thioester adduct. The thioester and the amide adducts are generated when GSH and GSSG react with oxanine-containing calf thymus DNA, and they might be possible forms of cellular DPCs. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should shed light on their detection in vivo and on their biological consequences.     

DNA-protein cross-links in different organs of mice induced by the combined action of zinc and gamma-irradiation

Fractional whole-body gamma-irradiation of mice at total doses of 0. 5-1.5 Gy induces increased DNA-protein cross-links (DPCs) in thymus, spleen, and brain, whereas in liver no DPCs are detected. Chronic administration of zinc ions in drinking water at concentration 10 mg/liter for 20-30 days increased DPCs in thymus, spleen, brain, and liver of mice. The combined action of zinc ions and gamma-radiation produced a significantly lower amount of DPCs than was induced by the separate action of these agents.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

Oxidatively induced DNA-protein cross-linking between single-stranded binding protein and oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.     

Interference by Cellular Melanin With Assay of DNA-Protein Crosslinks by the Potassium Dodecyl Sulfate Precipitation Method

The potassium dodecyl sulfate precipitation method was used to quantify DNA-protein crosslinks (DPCs) in lysates of melanoma cells exposed to ultraviolet radiation. Inducing melanin production in these cells before exposure to ultraviolet radiation decreased the apparent yield of DPCs. The decrease could also be produced by addition of melanin to lysates after exposure to crosslinking conditions. Experimental models could attribute this decrease to neither quenching of scintillations from the tritium label used nor to an effect of single strand breaks of DNA. This assay appears to be inappropriate for quantification of DPCs in melanized cells.     

Cold atmospheric-pressure plasma induces DNA–protein crosslinks through protein oxidation

Reactive oxygen and nitrogen species (ROS and RNS) generated by cold atmospheric-pressure plasma could damage genomic DNA, although the precise types of these DNA damage induced by plasma are poorly characterized. Understanding plasma-induced DNA damage will help to elucidate the biological effect of plasma and guide the application of plasma in ROS-based therapy. In this study, it was shown that ROS and RNS generated by physical plasma could efficiently induce DNA-protein crosslinks (DPCs) in bacteria, yeast, and human cells. An in vitro assay showed that plasma treatment resulted in the formation of covalent DPCs by activating proteins to crosslink with DNA. Mass spectrometry and hydroperoxide analysis detected oxidation products induced by plasma. DPC formation were alleviated by singlet oxygen scavenger, demonstrating the importance of singlet oxygen in this process. These results suggested the roles of DPC formation in DNA damage induced by plasma, which could improve the understanding of the biological effect of plasma and help to develop a new strategy in plasma-based therapy including infection and cancer therapy.     

Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta

Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1'-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase beta (polbeta). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polbeta. DPC formation was much less under repair than non-repair conditions, with extracts of either polbeta-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5'-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polbeta and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of >or=2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.     

DNA-protein cross-link formation mediated by oxanine. A novel genotoxic mechanism of nitric oxide-induced DNA damage

Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis.     

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 crosslinks as a biomarker of exposure to solar radiation: a preliminary study in brick-kiln workers.

In India, fired clay bricks are produced in small-scale factories. There are 60, 000 active brick kilns, providing employment to nearly 12 million people in different suboccupations. This industry is largely non-mechanized and operates from November to June. Almost all the workers are exposed to direct sunlight for 8-10 h a day. Cellular DNA-protein crosslinks (DPCs) are the biologically active nucleoprotein complexes formed between DNA and proteins. Ultraviolet light and gamma-rays, and other suspected carcinogens in humans, induce DPC formation in blood cells. DPCs have therefore been identified as a biomarker for monitoring exposure to these hazardous agents. Here we report steady-state levels of DPCs in human peripheral lymphocytes from 46 brick-kiln workers exposed occupationally for 8-10 h a day to solar radiation in brickfields and 25 unexposed controls. A significant increase (p <0.05) in DPC content and DPC coefficients in peripheral lymphocytes was observed in the brick-kiln workers compared with the controls. The data suggest that the DPC content of lymphocytes could be a possible biomarker of exposure to solar radiation. However, further work is necessary to confirm this.     

Photodynamic induction of DNA–protein cross-linking in solution by several sensitizers and visible light

The combined effect of several sensitizers and light on H ₂O or D₂O solutions of DNA-histone complexes, as well as the significance of singlet oxygen (¹O₂), in this photosensitizing reaction has been studied. On H₂O solutions, the production of ¹O₂, as well as the formation of DNA-protein cross-links (DPCs), were found to be dependent on light dose for all the sensitizers. Mesotetra (4N-methylpyridyl) porphine (T₄ MPyP), methylene blue (MB), and toluidine blue (TB) were the best photosensitizers with regard to tryptophan photolysis, followed by hematoporphyrin (HP), thioflavine T (TT), and pyronin G (PG). The formation of DPCs showed high initial rates, reaching a plateau at dose over 90 J/cm ². Under these irradiation conditions, the percentage of DPCs induced by the sensitizers decreases in the order T₄ MPyP > MB > TB ? HP ≈ TT ? PG (≈0). These DPCs were totally destroyed with proteinase K (15μg/ml). The irradiation of the DNA-histone-sensitizer solutions in the presence of L -carnosine (5 × 10^-4 M) produced approximately a 50% of DPCs inhibition for T₄ MPyP, MB, and TB, and a total inhibition for HP, TT, and PG. The substitution of H₂O by D₂O as solvent significantly increased the photodegradation of tryptophan, as well as the photoinduction of DPCs by the sensitizers. The results obtained indicate that singlet oxygen is the main agent responsible in the DNA–protein cross-linking formation. © 1993 John Wiley & Sons, Inc.     

Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells

DNA-protein cross-links (DPCs) present a formidable obstacle to cellular processes because they are "superbulky" compared with the majority of chemical adducts. Elimination of DPCs is critical for cell survival because their persistence can lead to cell death or halt cell cycle progression by impeding DNA and RNA synthesis. To study DPC repair, we have used DNA methyltransferases to generate unique DPC adducts in oligodeoxyribonucleotides or plasmids to monitor both in vitro excision and in vivo repair. We show that HhaI DNA methyltransferase covalently bound to an oligodeoxyribonucleotide is not efficiently excised by using mammalian cell-free extracts, but protease digestion of the full-length HhaI DNA methyltransferase-DPC yields a substrate that is efficiently removed by a process similar to nucleotide excision repair (NER). To examine the repair of that unique DPC, we have developed two plasmid-based in vivo assays for DPC repair. One assay shows that in nontranscribed regions, DPC repair is greater than 60% in 6 h. The other assay based on host cell reactivation using a green fluorescent protein demonstrates that DPCs in transcribed genes are also repaired. Using Xpg-deficient cells (NER-defective) with the in vivo host cell reactivation assay and a unique DPC indicates that NER has a role in the repair of this adduct. We also demonstrate a role for the 26 S proteasome in DPC repair. These data are consistent with a model for repair in which the polypeptide chain of a DPC is first reduced by proteolysis prior to NER.