Repair of Double-Strand Breaks in Bacteriophage T4 by a Mechanism That Involves Extensive DNA Replication

We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments. A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template. This reaction is characterized by the following interesting features. First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers. Second, repair of the dsb site is frequently associated with exchange of flanking DNA. Third, the repair reaction is absolutely dependent on the products of genes uvsX, uvsY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication. Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections. Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the SZOSTAK et al. model. We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair.     

Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.     

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G₀ fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10^–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.     

Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.     

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.     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.     

Possible occurrence of DNA double-strand breaks during repair of u.v.-induced damage in yeast

The yeast mutant rad54-3, which is temperature conditional for dsb rejoining, is sensitive to u.v. light when held at the restrictive temperature following exposure. We propose that this is attributable to the enzymatic formation of dsb in DNA containing u.v. lesions and a subsequent lack of dsb repair in this mutant.     

Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA.

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.     

Induction and repair of radiation-induced DNA double-strand breaks in human fibroblasts are not affected by terminal differentiation

It was studied for human skin fibroblasts, whether the induction or repair of DNA double-strand breaks (dsb) depend on the differentiation status. These studies were performed (a) with a fibroblast strain (HSF1) kept in progenitor state (mitotic fibroblasts, MF) or triggered to premature terminal differentiation (postmitotic fibrocytes, PMF) by exposure to mitomycin C or (b) with 20 fibroblast strains differing intrinsically in their differentiation status. The differentiation status was quantified by determining the fraction of postmitotic fibrocytes by light microscopy. DNA dsb were measured by constant-field gel electrophoresis, and the fraction of apoptotic cells by comet assay. MF and PMF cultures of HSF1 cells were irradiated with X-ray doses up to 160 Gy, and dsb were measured either immediately after irradiation or after a repair incubation of 4 or 24 h. There were a difference neither in the number of initial nor residual dsb. PMF cultures, however, showed a slightly higher number of dsb already present in non-irradiated cells, which was measured to result from a small fraction of 5% apoptotic cells. The 20 analysed fibroblast strains showed a substantial variation in the fraction of postmitotic fibrocytes (9-51%) as well as in the number of dsb remaining at 24 h after irradiation (1.9-4.9%), but there was no correlation between these two parameters. These data demonstrate that for fibroblasts the terminal differentiation has an effect neither on the induction nor the repair of radiation-induced dsb. This result indicates that the variation in dsb-repair capacity previously observed for fibroblast strains and which was considered to be the main cause for the variation in the cellular radiosensitivity, cannot be ascribed to differences in the differentiation status.     

Possible correlations between cell killing, chromosome damage and DNA repair after X-irradiation

To gain information about the possible pathway from primary DNA damage to cell killing via the formation of chromosome aberrations, we have examined the effects of the DNA synthesis inhibitor ara A on survival, on the occurrence of chromosome abnormalities and on the repair of DNA strand breaks. Our results are not inconsistent with the idea that the increased expression or 'fixation' of PLD measured after treatment with ara A is a reflection of an increase in the formation of chromosome damage comprising both exchange type and deletion type aberrations. These aberrations may arise from unrepaired or misrepaired dsb in the DNA. Treatment of irradiated cells with ara A results in a larger number of residual dsb which may be partly the reason for the increase in the frequency of acentric chromosome fragments. The reasons for the increase also in the frequency of exchange aberrations in the presence of ara A are not known but one possibility is that the probability of interaction between two dsb remains high during treatment with ara A due to the strong inhibition of dsb repair, whereas in untreated controls this probability decreases steeply with time after irradiation.     

Reduction of intracellular glutathione content and radiosensitivity

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or gamma-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced G2 delay.

Induction and repair of DNA double-strand breaks (dsb) was investigated in plateau phase Chinese hamster V79 cells and three radiosensitive mutant cell lines derived from them, irs-1, irs-2 and irs-3, using a pulsed-field gel electrophoresis assay, Asymmetric Field Inversion Gel Electrophoresis (AFIGE). There was no difference in the induction of DNA dsb per Gy and dalton between the radiosensitive mutant cells and wild-type V79 cells despite the wide differences in their radiosensitivity. Also, repair of DNA dsb proceeded in all cell lines with similar kinetics. In contrast to these observations at the DNA level, irradiation of exponentially growing cells showed a prolonged delay in G2 for irs-2 cells and a shortened delay in G2 for irs-1 cells, as compared to wild-type V79 cells. These results confirm previous observations suggesting that a deficiency in the rejoining of DNA dsb is unlikely to be the cause of the increased radiosensitivity of irs cells, and implicate alterations in postirradiation cell cycle progression as a possible cause for this phenomenon, although the mechanism is not known.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

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.     

Neutral filter elution detects only limited inhibition of double-strand break repair by 9-beta-D-arabinofuranosyladenine

Bradley and Kohn (1979) showed that the neutral filter elution technique detects DNA double-strand breaks (dsb), yet there is still some uncertainty regarding the interpretation of results obtained with this technique (viz. the curvilinear dose-response curve and the rapid repair kinetics). In this report we have investigated the effect of the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), a known inhibitor of dsb repair, on the DNA repair in X-irradiated Ehrlich ascites tumour cells as measured by this technique. We have compared the effect of ara A on repair in these cells with results previously obtained with the same cell line and using the neutral velocity sedimentation and DNA unwinding techniques (Bl?cher, 1982; Bryant and Bl?cher, 1982). Our results suggest that the lesions measured by the neutral elution technique are different from those measured by neutral velocity sedimentation or long-term repair measured by DNA unwinding.     

Interpretation of the dose and LET dependence of RBE values for lethal lesions in yeast cells

Survival data on yeast cells proficient or deficient in the repair of DNA double-strand breaks (dsb) and data on the induction of dsb are used to interpret the dose dependence of the RBE value for lethal lesions after irradiation at high dose rate followed by 72-hr liquid holding providing optimum conditions for repair of potentially lethal lesions (RBEDP, DP = delayed plating). The radiations applied are conventional (150 kV), soft (50 kV), and ultrasoft (4 kV) X rays, 30-MeV electrons (or 60Co gamma rays), and 3.5-MeV alpha particles. Analysis shows that the dose dependence of the RBEDP value can be explained by the combination of two dose-independent RBE values, one for the single-particle traversal effect (RBEspt) and the other for the accumulation of dsb (RBEdsb) due to the traversal of more than one particle through the cell nucleus. Furthermore, it is shown that the LET dependence of RBEspt values describing the linear component of the lethal lesions must be considered separately for "electron" and "particle" radiations.     

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

Relationship between double strand break rejoining and G2 block formation in V79 cells

Summary Caffeine and hypertonicity affect the survival of-irradiated Chinese Hamster V79 cells in different ways: while caffeine reduces the shoulder of the dose effect curve, hypertonic treatment mainly affects its final slope suggesting that different types of damage and (or) repair mechanisms are involved. Rejoining of DNA double strand breaks (dsb), as measured by neutral filter elution technique, exhibits a fast and a slow component, indicating that dsb rejoining consists of two different processes. Hypertonicity causes a temporary inhibition of the fast rejoining step but has no effect on the overall rejoining efficiency. Thus, it appears that its sensitizing effect on survival is not correlated with impaired dsb rejoining. Caffeine was found to inhibit the rejoining of dsb even after 6 h but the length of G2 phase was normal. By contrast, hypertonically treated cells are blocked in G2 but rejoining of dsb was normal. From these results we conclude that successful rejoining of part of the dsb involves arresting the cells reversibly in G2.Dedicated to Prof. G. Hotz on his 60^th birthday