Cross-links In Mammalian Cells Research Articles

Isolation and detection of DNA-protein crosslinks in mammalian cells.

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

Abstract 1579: Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate

Abstract Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induced base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-DNA cross links and DNA-protein cross-links in mammalian cells. We examined the ability of the soluble chromium compounds, sodium chromate (Na2CrO4), calcium chromate (CaCrO4) and potassium chromate (K2CrO4) to induce cytotoxicity and morphological transformation in C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III) intracellularly, making it a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. We hypothesized the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium chromate, and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate itself exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. We project that at concentrations of 0.1 mM and lower, ascorbate will enhance the cell transforming activity of Cr(VI) compounds. Supported by undergraduate fellowships from the Provost's Office at the University of Southern California (USC) (P. I., JRL), by funding from the M. S. Program in Molecular Microbiology and Immunology to JRL, by discretionary funding to JRL, and by prior grant ES03341 from NIEHS/NIH (P. I., JRL). Citation Format: Sophia A. Shahin, William Liao, Laureen Tran, Qasim A. Akinwumi, Farn-shuan Tseng, Alyssa Mathew-Joseph, Joseph R. Landolph. Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2014-1579

The Structure of the XPF-ssDNA Complex Underscores the Distinct Roles of the XPF and ERCC1 Helix- Hairpin-Helix Domains in ss/ds DNA Recognition

Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.

Homologous Recombination but Not Nucleotide Excision Repair Plays a Pivotal Role in Tolerance of DNA-Protein Cross-links in Mammalian Cells

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η

Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links.

Mutagenic potential of DNA–peptide crosslinks mediated by acrolein-derived DNA adducts

Current data suggest that DNA–peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA–protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA–peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA–peptide crosslinks in mammalian cells. Site-specific DNA–peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N 6 position of deoxyadenosine (dA) or the N 2 position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (γ-hydroxypropano-dA or γ-hydroxypropano-dG, respectively). In order to evaluate the potential of DNA–peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the γ-hydroxypropano-dA-mediated crosslink induced mutations at only ∼0.4%. In contrast, replication bypass of the γ-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of ∼8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at γ-hydroxypropano-dG caused an increase in mutation frequencies relative to γ-hydroxypropano-dG, similar modification of γ-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA–peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.

The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136.

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5′-GATC-3′ sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.

Arsenite induces oxidative DNA adducts and DNA-protein cross-links in mammalian cells

Arsenic is generally recognized as a nonmutagenic carcinogen because sodium arsenite induces DNA damage only at very high concentrations. In this study we demonstrate that arsenite concentrations above 0.25 μM induce DNA strand breaks in both human leukemia cells and Chinese hamster ovary cells. Therefore, DNA damage may be involved in arsenic-induced carcinogenesis. Formamidopyrimidine-DNA glycosylase and proteinase K greatly increased DNA strand breaks in arsenite-treated cells, providing evidence that a large portion of arsenite-induced DNA strand breaks come from excision of oxidative DNA adducts and DNA-protein cross-links. Because DNA strand breaks appear only temporarily during excision repair, the level of detectable DNA strand breaks will be low at any given time point. For this reason many previous studies have only detected low levels of DNA strand breaks. We also show that catalase, and inhibitors of calcium, nitric oxide synthase, superoxide dismutase, and myeloperoxidase, could modulate arsenite-induced DNA damage. We conclude that arsenite induces DNA adducts through calcium-mediated production of peroxynitrite, hypochlorous acid, and hydroxyl radicals.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Photobiological properties of 1-(3′-hydroxypropyl)-4,6,8-trimethylfuro[2,3- h]quinolin-2(1 H)-one, a new furocoumarin analogue

A new furoquinolinone derivative, 1-(3′-hydroxypropyl)-4,6,8-trimethylfuro[2,3- h]quinolin-2(1 H)-one (HPFQ, 4), was prepared, in which the nitrogen atom in position 1 carries a hydroxypropyl chain. The antiproliferative activity of HPFQ was studied in comparison with its analogue 1,4,6,8-tetramethylfuro[2,3- h]quinolin-2(1 H)-one (FQ) and 8-methoxypsoralen (8-MOP). By incubation in the dark, HPFQ, although retaining antitopoisomerase II activity, appeared less effective than FQ. Upon UVA irradiation, HPFQ produced little amounts of singlet oxygen, but detectable levels of superoxide anion; like FQ, HPFQ induced numbers of DNA–protein cross-links, but no interstrand cross-links in mammalian cells. The HPFQ phototoxicity was comparable to that of FQ and 8-MOP, while mutagenic activity, scored in two Escherichia coli strains, seemed much less remarkable.

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells.

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.

Defining the Roles of Nucleotide Excision Repair and Recombination in the Repair of DNA Interstrand Cross-Links in Mammalian Cells

Defining the Roles of Nucleotide Excision Repair and Recombination in the Repair of DNA Interstrand Cross-Links in Mammalian Cells

XK469, a selective topoisomerase IIbeta poison.

XK469 (NSC 697887) is a synthetic quinoxaline phenoxypropionic acid derivative that possesses unusual solid tumor selectivity and activity against multidrug-resistant cancer cells. We report here that XK469 and its S(-) and R(+)-isomers induce reversible protein-DNA crosslinks in mammalian cells. Under protein denaturing conditions, the protein-DNA crosslinks are rendered irreversible and stable to DNA banding by CsCl gradient ultracentrifugation. Several lines of evidence indicate that the primary target of XK469 is topoisomerase IIbeta. Preferential targeting of topoisomerase IIbeta may explain the solid tumor selectivity of XK469 and its analogs because solid tumors, unlike leukemias, often have large populations of cells in the G(1)/G(0) phases of the cell cycle in which topoisomerase IIbeta is high whereas topoisomerase IIalpha, the primary target of many leukemia selective drugs, is low.

Formation of DNA-protein cross-links in mammalian cells by levuglandin E2.

Levuglandin E2 (LGE2), a rearrangement product derived from the prostaglandin endoperoxide, PGH2, causes repair-resistant DNA-protein cross-links and cell death (LD50 = 230 nM) in V79 Chinese hamster lung fibroblasts. The half-life for sequestration of LGE2 by covalent binding to cellular nucleophiles is at least an hour for 10 microM LG. This suggests that the in vivo production and distribution of free LGs should be measurable on this time scale. Following removal of the LGE2 and the return of the cultures to normal growth medium, additional DNA-protein cross-links continued to form over the ensuing 6-24 h. The results suggest that LG adducts to DNA or protein are not repaired, but react further at sites on protein or DNA in close proximity to the initial adducts, forming cross-links in a slow phase of the process.

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.

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)diamminedichloride 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 μM 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: (A) 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. (B) Either “used” culture medium or cycloheximide (1 μg/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01–5 μg/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. (C) 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. (D) In the presence of aphidicolin (10 μg/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. (E) Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled DNA indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of DNA 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 linked with cell cycling.

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) ( cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), l-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37°C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and l-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.

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 m m sodium hydroxide (0.33 ml/min) instead of the standard 20 m m 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 μ m, 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.

Repair of deoxyribonucleic acid lesions caused by adriamycin and ellipticine

The intercalating agents adriamycin and ellipticine caused DNA single and double strand breaks and DNA-protein crosslinks in mammalian cells. Ellipticine caused a much higher frequency of each of these lesions than adriamycin, at equitoxic doses. However, adriamycin-induced DNA effects were slowly and incompletely repaired over 24 hr after drug removal while the effects of ellipticine were virtually completely repaired within 30 min. Repair of ellipticine-induced lesions was observed only if cells were diluted (~100-fold) after drug treatment, suggesting that significant amounts of drug may have been sequestered in the cellular membranes. Cell dilution did not alter the effects of adriamycin. Removal of the DNA lesions was temperature dependent. Finally, the similarity in the rates of repair for DNA single and double strand breaks and DNA-protein crosslinks supports the hypothesis that they are functionally related.