Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.     

Repair of DNA single-strand and double-strand breaks in the Chinese hamster xrs 5 mutant cell line as determined by DNA unwinding

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray-sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.     

Repair of DNA double-strand breaks in colcemid-arrested mitotic Chinese hamster cells

Chinese hamster V79 cells blocked in mitosis were irradiated with 60Co gamma-rays and incubated for repair in the presence of colcemid. DNA strand breaks were measured using neutral sucrose gradient centrifugation or the alkaline unwinding technique. It was found that mitotic cells repair DNA double-strand breaks (as well as single-strand breaks) efficiently, with a rate similar to exponentially growing asynchronous cells. It is argued that the dense packing of the chromatin in the mitotic chromosome makes a recombinational repair mechanism unlikely.     

Elevated levels of DNA double-strand breaks (dsb) in restriction endonuclease-treated xrs5 cells correlate with the reduced capacity to repair dsb.

Recently we have reported the kinetics of DNA double-strand breaks (dsb) induced in electroporated mammalian (CHO) cells that had been treated with the restriction endonuclease PvuII, as measured by the filter elution assay at the non-denaturing pH of 9.6. A gradual accumulation of dsb was observed over a 24-h incubation period following the restriction endonuclease (RE) treatment and this was attributed to a competition between incision of the DNA by PvuII and dsb repair. In order to test this 'competition' hypothesis we have carried out similar experiments in the radiosensitive xrs5 mutant cell line, which has been shown to be deficient in dsb repair. The levels of dsb monitored by the non-denaturing filter elution assay in the xrs5 cell line treated with PvuII was found to be 3-4 times higher than that found for the wild-type CHO K1 cell line. Levels of dsb were also significantly raised in xrs5 cells treated with BamHI, as compared with the background levels observed in the CHO line. These data lend strong support to the competition hypothesis of simultaneous incision and repair of RE-induced dsb.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Induction of DNA double-strand breaks in Chinese hamster V79 cells by 2-chlorodeoxyadenosine

2-Chlorodeoxyadenosine was found to induce DNA double-strand breaks as well as cell death in log-phase Chinese hamster V79 cells. The induction of DNA double-strand breaks, measured by a neutral elution technique, was observed after a 2-h incubation of the cells in the presence of 5 microM of 2-chlorodeoxyadenosine, but these breaks were almost rejoined by a subsequent 1-h incubation, even though this drug was present in the medium during incubation. This repair was prevented by the addition of nicotinamide, which is known to inhibit poly(ADP-ribose) synthesis that is strongly associated with the DNA ligation, but not prevented by the addition of 9-beta-D-arabinofuranosyladenine (araA), which is known to inhibit DNA polymerization. These results suggest that the repair of CdA-induced double-strand breaks is achieved by ligation alone without DNA polymerization. When 35 microM of cycloheximide and 1.3 mM of dibutyryl cAMP were added to the medium, it was found that the induction of double-strand breaks by 2-chlorodeoxyadenosine was suppressed, while the cytotoxicity of 2-chlorodeoxyadenosine measured by colony-forming ability was not interfered with. These results suggest that the induction of DNA double-strand breaks is not associated with the cytotoxicity of this drug.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of DNA double-strand breaks (DSBs] by restriction endonucleases (REs) and inhibitors of DNA topoisomerase II known to induce DNA strand breaks. Five different types of REs, namely CfoI, EcoRI, HpaII (which induce cohesive DSBs), HaeIII and AluI (which induce blunt DSBs) were employed. REs that induce blunt-end DNA DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks. xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type CHO-K1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant CHO cells, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type cells. We also studied the effect of inhibitors of DNA topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type CHO-K1 cells respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G1 treatment of CHO cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than CHO-K1 cells. The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of DNA topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.     

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.     

Biochemical mechanisms of chromosomal translocations resulting from DNA double-strand breaks

Exposure of mammalian cells to agents that induce DNA double-strand breaks typically results in both reciprocal and nonreciprocal chromosome translocations. Over the past decade, breakpoint junctions of a significant number of translocations and other genomic rearrangements, both in clinical tumors and in experimental models, have been analyzed at the DNA sequence level. Based on these data, reasonable inferences regarding the biochemical mechanisms involved in translocations can be drawn. In a few cases, breakpoints have been shown to correlate with sites of double-strand cleavage by agents to which the cells or patients have been exposed, including exogenous rare-cutting endonucleases, radiomimetic compounds, and topoisomerase inhibitors. These results confirm that translocations primarily reflect misjoining of the exchanged ends of two or more double-strand breaks. Many junctions show significant loss of DNA sequence at the breakpoints, suggesting exonucleolytic degradation of DNA ends prior to joining. The size and frequency of these deletions varies widely, both between experimental systems, and among individual events in a single system. Homologous recombination between repetitive DNA sequences does not appear to be a major pathway for translocations associated with double-strand breaks. Rather, the general features of the junction sequences, particularly the high frequency small terminal deletions, the apparent splicing of DNA ends at microhomologies, and gap-filling on aligned double-strand break ends, are consistent with the known biochemical properties of the classical nonhomologous end joining pathway involving DNA-dependent protein kinase, XRCC4 and DNA ligase IV. Nevertheless, cells with deficiencies in this pathway still exhibit translocations, with grossly similar junction sequences, suggesting an alternative but less conservative end joining pathway. Although evidence for participation of specific DNA end processing enzymes in formation of translocations is largely circumstantial, likely candidates include DNA polymerases lambda and mu, Artemis nuclease, polynucleotide kinase/phosphatase, tyrosyl-DNA phosphodiesterase, DNase III, Werner syndrome protein, and the Mre11/Rad50/NBS1 complex.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by the restriction endonuclease Dra I: dose-effect relationships and effect of substitution of chromosomal DNA with bromodeoxyuridine

Treatment of G1-phase Chinese hamster ovary (CHO) cells with the restriction endonuclease Dra I (recognition site TTT/AAA) leads to the induction of chromosome-type aberrations. The dose-effect relationships or the frequencies of polycentric chromosomes have a strong linear component. Prelabelling of the cells with bromodeoxyuridine (B) leads to a strong suppression by the chromosome breaking activity of Dra I. This may be explained by assuming that substitution of T by B renders the recognition site of Dra I resistant to being cut by the enzyme.     

Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells

The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G₂ than in G₁, suggesting that DSB rejoining in G₂ proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G₁ and G₂ compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G₂ occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G₁ and G₂. We propose that structural characteristics of G₂ phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.     

Evidence that DNA double-strand breaks initiate the phenotype of delayed reproductive death in Chinese hamster ovary cells.

A persistently reduced cloning efficiency occurs in many of the cloned progeny of Chinese hamster ovary (CHO) cells surviving X irradiation, a stable phenotype we have previously termed delayed reproductive death (Int. J. Radiat. Biol. 60, 483-496, 1991). We now report that this phenotype is also induced by the alkylating agent ethyl methanesulfonate (EMS), but not by irradiation with ultraviolet light. The restriction endonuclease HinfI, which binds at G [symbol: see text] ANTC DNA sequences and generates cohesive-end double-strand breaks, was also efficient in inducing delayed reproductive death. On the other hand, an X-ray-sensitive CHO mutant, xrs-5, which is defective in the repair of DNA double-strand breaks, did not show this phenotype following X irradiation. These results suggest that DNA double-strand breaks, as well as the endogenous repair processes for these breaks, are associated with the induction of the delayed reproductive death phenotype in CHO cells. The possible mechanism for the induction of delayed reproductive death by EMS is discussed.     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

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.     

The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.