检测DNA-蛋白质交联的新方法

将彗星实验进行改进以用于DNA-蛋白质交联作用的检测。利用甲醛对受试动物肝细胞的影响来判定此法是否适用于检测DNA-蛋白质交联。由于在实验中添加一定量的蛋白酶K, 可使单细胞在电泳时产生更大的迁移, 因此可以利用添加蛋白酶K前后的彗星尾距比来判断外来化合物对生物机体产生DNA损伤效应的时候是否有出现DNA-蛋白质交联作用。结果表明, 该方法快速、经济、灵敏度较高, 可以在单细胞水平对甲醛等强交联剂引发的不同组织的DNA-蛋白质交联效应进行检测, 希望该方法能成为指示DNA交联能力的有用工具。     

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.     

Repair pathway for PARP-1 DNA-protein crosslinks

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.     

Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay

Oxaliplatin is frequently used in the therapy of cancer. In DNA, oxaliplatin induces, like cisplatin, the formation of crosslinks, which are commonly accepted as being responsible for the cytotoxicity of platinum agents. The detection of oxaliplatin-induced DNA crosslink formation and repair could be a good measure of assessing how a patient is responding to the agent. In this study, we used a validated modification of the alkaline comet assay for detecting the presence of these crosslinks in vitro and in cancer patients. The H460 tumour cell line was treated in vitro with a range of oxaliplatin and cisplatin doses, and the subsequent crosslink formation and repair compared between the two agents. In addition, lymphocytes from cancer patients undergoing oxaliplatin-based chemotherapy were assayed for the formation and repair of oxaliplatin-induced crosslinks. A dose-response was observed in the in vitro samples, with cisplatin producing more crosslinks than oxaliplatin at equimolar concentrations and lesions induced by both agents showing different repair efficiencies. Furthermore, evidence of crosslink formation and repair was observed in the peripheral blood lymphocytes of all cancer patients studied, along with the detection of interindividual variability in crosslink formation and repair efficiencies. To the best of our knowledge, this is the first time that oxaliplatin DNA crosslinks have been detected either in vitro or in patient samples using the alkaline comet assay. Due to its sensitivity, rapidity, small cell sample and low cost, the alkaline comet assay is a good method for the detection of oxaliplatin-induced crosslinks and their subsequent repair and, in future clinical studies, could prove to be a valuable tool in assessing/predicting a patient's response to chemotherapy.     

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.     

Formation and repair of DNA-protein crosslinks in newly replicated DNA

The production and removal of gamma-radiation-induced DNA-protein crosslinks (DPC) in nuclear matrix-associated newly replicated DNA were examined, as well as the relationship of DPC to DNA replication. In unirradiated, exponentially growing Chinese hamster V79 cells, DNA pulse labeled with [3H]thymidine was observed to be bound preferentially to protein. The pulse-labeled DNA subsequently became dissociated from protein. After a 30- to 60-min chase period, the level of labeled DNA in DPC was reduced to the same level as for bulk DNA. The radiation dose response for the formation of DPC was similar in newly replicated DNA that had been chased for various times and in mature chromatin DNA. Labeled DNA, in the DPC formed after 60 Gy, was rapidly removed from protein during the postirradiation incubation period. However, no recovery of DNA synthesis was observed, even after the majority of DPC were released. Thus either DPC are not the sole cause of the inhibition of DNA synthesis or their removal is not sufficient for DNA synthesis to resume.     

The FANCJ helicase unfolds DNA-protein crosslinks to promote their repair

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High-LET irradiation of a DNA-binding protein: protein-protein and DNA-protein crosslinks

The chromosomal protein MC1 is a monomeric protein of 93 amino acids that is able to bind any DNA but has a slight preferential affinity for some sequences and structures, like cruciform and minicircles. The protein has been irradiated with 36Ar18+ ions of 95 MeV/nucleon. The LET of these particles in water is close to 270 keV/microm. We tested the activity of the protein by measuring its ability to form complexes with DNA. We tested the integrity of the protein by measuring the molecular weight of the species formed. Compared with gamma radiation, we observed for the same dose a less efficient inactivation of the protein, a greater protection of the protein by the bound DNA, a lower induction of chain breakage, and a greater production of protein-protein and DNA-protein crosslinks. The results are discussed in terms of the quantitative and the qualitative differences between the two types of radiation: The global radical yield is slightly higher with gamma rays, whereas the density of radicals produced along the particle track is considerably higher with argon ions.     

Increased DNA-protein crosslinks in lymphocytes of residents living in chromium-contaminated areas

It has been known for a number of years that chromium-containing mine slags were used as landfill in residential areas of Hudson County, New Jersey. Since one of the major lesions induced in intact cells by chromate is the DNA-Protein crosslink, we have used this lesion as a biomarker of biological effect of chromium (Cr) exposure. We have previously developed a sensitive and easy-to-perform assay to detect DNA-Protein crosslinks, based on the selective K SDS precipitation of DNA associated with protein. We examined the levels of DNA-Protein crosslinks in peripheral blood mononuclear cells of 33 individuals determined to be at risk for chromium exposure by virtue of their residence in Hudson County and their urinary Cr levels. These data were compared to the levels of DNA-Protein crosslinks among 49 controls who resided in noncontaminated areas. A complete clinical examination and urine analysis did not show any Cr-related abnormalities among the exposed population. The mean DNA-Protein crosslink level in the lymphocytes of the exposed group was 1.3±0.5% (SD), whereas the unexposed group had 0.8±0.4% (p<0.001), after adjustment for age, gender, race, smoking, and weight. Further studies in this population are needed to confirm the possible association between the high levels of DNA-Protein crosslink and Cr exposure.     

A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis

The covalent crosslinking of protein to DNA is a form of DNA damage induced by a number of commonly encountered agents, including metals, aldehydes, and radiation as well as chemotherapeutic drugs. DNA-protein crosslinks (DPCs) are potentially bulky and helix distorting and have the potential to block the progression of translocating protein complexes. To fully understand the induction and repair of these lesions, it will be important to identify the crosslinked proteins involved. To take advantage of dramatic improvements in instrument sensitivity that have facilitated the identification of proteins by proteomic approaches, improved methods are required for isolation of DPCs. This article describes a novel method for the isolation of DPCs from mammalian cells that uses chaotropic agents to isolate genomic DNA and stringently remove noncrosslinked proteins followed by DNase I digestion to release covalently crosslinked proteins. This method generates high-quality protein samples in sufficient quantities for analysis by mass spectrometry. In addition, the article presents a modified form of this method that also makes use of chaotropic agents for promoting the adsorption of DNA (with crosslinked proteins) to silica fines, markedly reducing the DPC isolation time and cost. These approaches were applied to radiation- and camptothecin-induced DPCs.     

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.     

DNA-protein crosslinks: their induction, repair, and biological consequences

The covalent crosslinking of proteins to DNA presents a major physical challenge to the DNA metabolic machinery. DNA-protein crosslinks (DPCs) are induced by a variety of endogenous and exogenous agents (including, paradoxically, agents that are known to cause cancer as well as agents that are used to treat cancer), and yet they have not received as much attention as other types of DNA damage. This review summarizes the current state of knowledge of DPCs in terms of their induction, structures, biological consequences and possible mechanisms of repair. DPCs can be formed through several different chemistries, which is likely to affect the stability and repair of these lesions, as well as their biological consequences. The considerable discrepancy in the DPC literature reflects both the varying chemistries of this heterogeneous group of lesions and the fact that a number of different methods have been used for their analysis. In particular, research in this area has long been hampered by the inability to chemically define these lesions in intact cells and tissues. However, the emergence of proteomics as a tool for identifying specific proteins that become crosslinked to DNA has heralded a new era in our ability to study these lesions. Although there are still many unanswered questions, the identification of specific proteins crosslinked to DNA should facilitate our understanding of the down-stream effects of these lesions.     

DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds

Abstract

DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds. Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas. The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects. The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure. Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers. Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DNA adducts were saturated at maximum levels. The saturation of DNA-protein crosslinks seems to occur at 7–8 μg I-¹ chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 μg l^?1. Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes. The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls. Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age or weight.     

Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks

Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single-strand break intermediates and without a detectable accumulation of double-strand breaks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies.     

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3′-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (“long-patch”) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5′-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15–30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60–70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5′-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.     

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.     

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.     

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.