Detection of heavy-ion-induced DNA double-strand breaks using static-field gel electrophoresis

Radiation-induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up, and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6–7 keV/µm), while for high energy oxygen ions (20keV/µm) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET=250 keV/µm) were found to show RBEs substantially below unity, and for higher LET particles (31 y-250 keVµm) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but rather misrepaired or unrepaired DNA lesions are related to cellular inactivation.     

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.     

Randomly distributed DNA double-strand breaks as measured by pulsed field gel electrophoresis: A series of explanatory calculations

The aim of this article is to characterize expressions of relevance to the interpretation of pulsed field gel electrophoresis (PFGE) experiments where randomly distributed double-strand breaks (DSBs) are detected as smears of DNA fragments. Specifically, equations for conversion of percentages of fragments in defined size ranges to DSBs were derived. Several models have been used, one of which is based on theoretically fragmented DNA from the fission yeastSchizosaccharomyces pombe, which has three PFGE separable chromosomes.     

Cystic fibrosis: screening for a DNA deletion by field inversion gel electrophoresis

Summary We analyzed DNA of ten cystic fibrosis (CF) patients representing DNA of 19 different CF chromosomes and screened for deletions by means of field inversion gel electrophoresis (FIGE). No differences were detected after digestion of the DNA samples with the restriction enzymes Not I and Sfi I and hybridization with the probes MetH, MetD, J3.11, and 7C22. Thus the percentage of deletions occurring within the CF region and detectable with FIGE is less than 15.2% (95% confidence interval, N=19)     

Combination of static-field gel electrophoresis and densitometric scanning for the determination of radiation-induced DNA double-strand breaks in CHO cells

An experimental setup using static-field gel electrophoresis (SFGE) was developed to determine radiation-induced DNA double-strand breaks (DSBs) in CHO-K1 cells after exposure to X-rays or heavy charged particles. The fraction of DNA eluted into the gel matrix depends on the quantity of DSBs introduced. In agreement with a recent report, SFGE and pulsed-field electrophoresis were found to be equally sensitive in DSB detection. With radiolabeled DNA from cell cultures, the absolute amount of DNA migrating out of agarose plugs into the gel was quantified by determining the radioactivity in the gel lane. Alternatively, relative measurements of the amount of DNA released into the gel were achieved with a standardized protocol for both SFGE and a subsequent densitometric scanning of photographic negatives from gels stained with ethidium bromide. After calibration with the radioactive method, the fractions of DNA retained could be calculated directly from the data obtained with the densitometric assay to set up classical dose-effect curves. This procedure was validated for its application with heavy ions using an 500 MeV/u lead beam.     

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.     

Analysis of restriction enzyme-induced DNA double-strand breaks in Chinese hamster ovary cells by pulsed-field gel electrophoresis: implications for chromosome damage.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

X-ray-induced DNA double-strand breaks in mouse l1210 cells: a new computational method for analyzing neutral filter elution data

The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape.     

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Secondary pulsed field gel electrophoresis: a new method for faster separation of larger DNA molecules.

A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols.     

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.     

Positional stability of single double-strand breaks in mammalian cells

Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNA-end binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.     

Capture of DNA sequences at double-strand breaks in mammalian chromosomes

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.     

Involvement of DNA polymerase mu in the repair of a specific subset of DNA double-strand breaks in mammalian cells

The repair of DNA double-strand breaks (DSB) requires processing of the broken ends to complete the ligation process. Recently, it has been shown that DNA polymerase μ (polμ) and DNA polymerase λ (polλ) are both involved in such processing during non-homologous end joining in vitro. However, no phenotype was observed in animal models defective for either polμ and/or polλ. Such observations could result from a functional redundancy shared by the X family of DNA polymerases. To avoid such redundancy and to clarify the role of polμ in the end joining process, we generated cells over-expressing the wild type as well as an inactive form of polμ (polμD). We observed that cell sensitivity to ionizing radiation (IR) was increased when either polμ or polμD was over-expressed. However, the genetic instability in response to IR increased only in cells expressing polμD. Moreover, analysis of intrachromosomal repair of the I-SceI-induced DNA DSB, did not reveal any effect of either polμ or polμD expression on the efficiency of ligation of both cohesive and partially complementary ends. Finally, the sequences of the repaired ends were specifically affected when polμ or polμD was over-expressed, supporting the hypothesis that polμ could be involved in the repair of a DSB subset when resolution of junctions requires some gap filling.     

Studies on mammalian mutants defective in rejoining double-strand breaks in DNA

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.     

Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in S-phase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature S-phase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ?H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ?H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ?H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.     

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5′ overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.