Formation of strand breaks and interstrand cross-links in DNA by methylglyoxal

Methylglyoxal (MG), a dietary mutagen, is present in various frequently consumed beverages and foods and in cigarette smoke. A combination of S1 nuclease hydrolysis and alkaline unwinding assay was used to demonstrate the formation of single-strand breaks and interstrand cross-links in DNA upon treatment with MG. Calf thymus DNA, when treated with increasing concentrations of MG, showed an increasing degree of S1 nuclease hydrolysis. It also showed the formation of an increasing number of strand breaks per molecule as determined by an alkaline unwinding assay. Incubation of DNA with relatively higher concentrations of methylglyoxal or prolonged treatment gave increased thermal melting temperatures and an enhanced rate of reannealing after thermal denaturation. These results indicated the formation of interstrand cross-links. Upon treatment with MG, A-T base pair depleted DNA showed a reduced number of single-strand break formation. It also showed a significantly lower decrease in Tm as compared with MG-treated normal DNA. These results showed that under the conditions used, MG primarily reacts with A-T base pairs in duplex DNA.     

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.     

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.     

Induction of multiple plasmid recombination in Saccharomyces cerevisiae by psoralen reaction and double strand breaks.

DNA damage-induced multiple recombination was studied by cotransforming yeast cells with pairs of nonreplicating plasmids carrying different genetic markers. Reaction of one of the plasmids with the interstrand crosslinking agent, psoralen, stimulated cellular transformation by the undamaged plasmid. The cotransformants carried copies of both plasmids cointegrated in tandem arrays at chromosomal sites homologous to either the damaged or the undamaged DNA. Plasmid linearization, by restriction endonuclease digestion, was also found to stimulate the cointegration of unmodified plasmids. Disruption of the RAD1 gene reduced the psoralen damage-induced cotransformation of intact plasmid, but had no effect on the stimulation by double strand breaks. Placement of the double strand breaks within yeast genes produced cointegration only at sequences homologous to the damaged plasmids, while digestion within vector sequences produced integration at chromosomal sites homologous to either the damaged or the undamaged plasmid molecules. These observations suggest a model for multiple recombination events in which an initial exchange occurs between the damaged DNA and homologous sequences on an undamaged molecule. Linked sequences on the undamaged molecule up to 870 base pairs distant from the break site participate in subsequent exchanges with other intact DNA molecules. These events result in recombinants produced by reciprocal exchange between three or more DNA molecules.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

Coping with DNA double strand breaks

The repair of DNA double strand breaks (dsb) is important for maintaining the physical and genetic integrity of the genome. Moreover, in humans it is associated with the prevention of diseases such as immune deficiencies and cancer. This review briefly explores the fundamental strategies for repairing dsb, examines how cells maximize the fidelity of dsb repair in the cell cycle and discusses the requirements for dsb repair in the context of chromatin.     

Phased psoralen cross-links do not bend the DNA double helix

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies base on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5 degrees [Pearlman, D. A., Holbrook, S. R., Pirkle, D. H., & Kim, S.-H. (1985) Science (Washington, D.C.) 227, 1304-1308]. On the other hand, Sinden and Hagerman [Sinden, R. R., & Hagerman, P. J. (1984) Biochemistry 23, 6299-6303] find that, in solution, psoralen cross-linked DNA is not bent. Here we use gel electrophoresis to test the validity of the current models. We have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28 degrees unwinding of the helix by HMT [Wiesehahn, G., & Hearst, J. E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2703-2707], one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. We find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. Our observations place an upper limit of 10 degrees on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.     

Repair of DNA interstrand cross-links

DNA interstrand cross-links (ICLs) are very toxic to dividing cells, because they induce mutations, chromosomal rearrangements and cell death. Inducers of ICLs are important drugs in cancer treatment. We discuss the main properties of several classes of ICL agents and the types of damage they induce. The current insights in ICL repair in bacteria, yeast and mammalian cells are reviewed. An intriguing aspect of ICLs is that a number of multi-step DNA repair pathways including nucleotide excision repair, homologous recombination and post-replication/translesion repair all impinge on their repair. Furthermore, the breast cancer-associated proteins Brca1 and Brca2, the Fanconi anemia-associated FANC proteins, and cell cycle checkpoint proteins are involved in regulating the cellular response to ICLs. We depict several models that describe possible pathways for the repair or replicational bypass of ICLs.     

Repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. If not repaired they can lead to cell death. If misrepaired DSBs contribute to chromosomal aberrations and genomic instability. Non-homologous end joining (NHEJ) is one of two major pathways for the repair of DSBs in human cells. Proteins known to be required for NHEJ include the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis. This review discusses how these and other accessory proteins may function in the repair of DSBs produced by ionizing radiation (IR) and by V(D)J recombination.     

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.     

In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1-100 microM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 microM we observed an increase in the tail length, whereas at 100 microM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA-DNA and/or DNA-protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 microM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA-protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells

DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.     

Diepoxybutane interstrand cross-links induce DNA bending

The bifunctional alkylating agent 1,2,3,4-diepoxybutane (DEB) is thought to be a major contributor to the carcinogenicity of 1,3-butadiene, from which it is derived in vivo. DEB forms DNA interstrand cross-links primarily between distal deoxyguanosine residues at the duplex sequence 5′-GNC. In order for the short butanediol tether to span this distance, distortion of the DNA target has been postulated. We determined that the electrophoretic mobility of ligated DNA oligomers containing DEB cross-links was retarded in comparison with control, uncross-linked DNA. Our data are consistent with DNA bending of ∼34° per lesion towards the major groove.     

A nervous predisposition to unrepaired DNA double strand breaks

Ataxia-telangiectasia (A–T) has for a long time stood apart from most other human neurodegenerative syndromes by the characteristic failure of cells derived from these patients to properly repair DNA damage-induced by ionizing radiation. The discovery of mutations in the ATM gene as being the underlying cause for A–T and the demonstration that the ATM protein functions as a DNA damage-responsive kinase has defined current research focusing on decoding how the cell responds to genotoxic stress. Yet, despite significant advances in delineating the cellular DNA damage response pathways coordinated by ATM, very little headway has been made toward understanding how loss of ATM leads to progressive cerebellar ataxia and whether this can be attributed to an underlying defect in DNA double strand break repair (DSBR). Since its identification, A–T has been used as the archetypal model for how a deficiency in DNA repair affects both the development and maintenance of the nervous and immune systems in humans as well as contributing to the process of tumourigenesis. However, following the growing availability and cost effectiveness of next generation sequencing technologies, the increasing recognition of novel human disorders associated with abnormal DNA repair has demonstrated that the neuropathology typified by A–T is an ‘exception’ rather than the ‘rule’. As a consequence, this throws into doubt the longstanding hypothesis that the neurodegeneration seen in A–T is due to the progressive loss of damaged neurons that have acquired toxic levels of unrepaired DNA lesions over time. Therefore, this review aims to address the question: Is defective DNA double strand break repair an underlying cause of neurodegeneration?