Rejoining of double strand breaks in normal human and ataxia-telangiectasia fibroblasts after exposure to 60Co gamma-rays, 241Am alpha-particles or bleomycin

The rejoining of DNA double strand breaks (dsb) induced by 60Co gamma-rays, 241Am alpha-particles or bleomycin was measured by neutral filter elution. In agreement with their colony-forming ability, ataxia-telangiectasia cells (AT2BE) and normal fibroblasts exhibited similar dsb rejoining capacity following alpha-irradiation, but showed marked differences in the rejoining kinetics of dsb induced by gamma-rays or bleomycin.     

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.     

Acceleration of DNA-double strand rejoining during the adaptive response of Chlamydomonas reinhardtii

Newly developed constant-field low voltage electrophoresis (adapted for algae cells by us) was applied to quantify the induction and repair of nuclear DNA double-strand breaks, by measuring the movement of DNA out of the starting wells into the electrophoresis gel using a UV-gel scan and computer analysis of DNA-ethidium bromide fluorescense (Syngene; Gene tools). A cell-wall-less mutant strain of Chlamydomonas reinhardtii (CW15) was used; the DNA and proteins are easily accessible because of the lack of an outer cell wall. Our results showed that giving a small priming dose (50 Gy) led to a small acceleration of dsb rejoining. When the magnitude of the priming dose was progressively increased, there was a corresponding decrease in the fraction of damage remaining at 4 hours after radiation exposure (to a test dose of 500 Gy). This indicates an upregulated rejoining of dsb following exposure of cells to the priming dose, which may be related to the strong adaptive response in this organism. Protein synthesis inhibitors were found to reduce the rate of rejoining of dsb, and from earlier results are known to inhibit the adaptive response. Thus, the adaptive response is likely to be dependent on increased dsb rejoining and depends on de novo protein synthesis. The nature of these proteins has not yet been established. C. reinhardtii CW15 is an attractive model system in which to study the underlying mechanisms of the adaptive response to ionizing radiation, and its underlying link with dsb rejoining. The results are interesting both from a basic biological point of view, and as a means to further understand the response of tumour cells to radiation therapy since the adaptive response has been postulated to determine the shape of the "shoulder" region of the survival curve of cells at low doses of radiation.     

Comparative study of G2 delay and survival after241Americium-α and60Cobalt-γ irradiation

Summary Survival and G2 delay following exposure to either⁶⁰Cobalt--rays or²⁴¹Americium--paticles were studied in eight mammalian cell lines of human and animal origin including human fibroblasts from normal individuals and from patients with Ataxia telangiectasia or Fanconi's anemia.For both endpoints the effectiveness of alpha particles was greater as compared to-rays. RBE values for G2 delay (4.6–9.2) were in general comparable to RBE values derived from initial slopes of survival curves (RBE ) but higher compared to the ratio of mean inactivation doses .Ataxia cells were particularly sensitive to cell killing by-irradiation (D₃₇ = 0.57 Gy), however, showed average sensitivity to-particles of high LET (D₃₇ = 0.30 Gy).With the exception of Ataxia cells, cell killing and G2 delay seem to be related processes if individual cell cycle parameters are taken into account.     

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.     

Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radiosensitivity

Five recently established cell lines of human carcinoma of the cervix of varying radiosensitivity have been used to determine whether the induction or rejoining of DNA double-strand breaks (dsb) shows any correlation with radiosensitivity or radiation recovery capacity. Double-strand DNA breaks have been measured using neutral filter elution at pH 9.6. The number of breaks induced immediately after irradiation with doses of 10 to 40 Gy 60Co gamma rays appeared to show some correlation with radiosensitivity particularly after 10 Gy; the two more radiosensitive lines incurred more breaks than the more radioresistant lines. In addition, the shape of the induction curve with dose was linear for the two sensitive lines but curvilinear for the resistant lines. Despite the dose scales being different, this mirrored their respective cell survival curve shapes. After 30 or 50 Gy irradiation, rejoining of breaks appeared to be rapid and almost complete within 60 min at 37 degrees C for the three resistant lines. However, for the sensitive lines, one line (HX160c) in particular exhibited a reduced rate of dsb rejoining. In addition, a residual level of dsb was present in this line even after allowing rejoining for 3 h. While induction and rejoining of DNA dsb therefore appears to be a factor in determining radiosensitivity, at doses relevant to cellular survival (up to 10 Gy), the greater induction of DNA dsb in radiosensitive lines may play a significant role in determining the cellular response to ionizing radiation.     

Hydrogen peroxide lethality is associated with a decreased ability to maintain positive DNA supercoiling

Hydrogen peroxide is more toxic to mammalian cells at 37 degrees C than 0 degree C at all concentrations studied. Histone-free nuclei (nucleoids) extracted from treated cells have a reduced ability to maintain positive DNA supercoiling, with the maximum effect at the higher temperature. Prior exposure of cells to sodium ascorbate at 0 degree C increased both toxicity and the inhibition of nuclear supercoil rewinding. After exposure at 0 degrees C, normal levels of supercoiling returned with both a fast and a slow component, kinetics characteristic of DNA single-strand break repair; the fast component was eliminated when cells were exposed at 37 degrees C due to in situ rejoining. At least a portion of the lethal lesions induced by hydrogen peroxide are DNA double-strand breaks (dsb) because the dsb repair-deficient mutant, xrs-5, is approximately two to three times more sensitive than wild-type cells over the initial portion of the survival curve. However, the increased toxicity found after exposure at 37 degrees C is observed equally in both cell lines, indicating that temperature-dependent cell killing is not directly linked to DNA dsb. It is suggested that cell killing at 37 degrees C is mediated through two linked processes. First, hydrogen peroxide may disrupt cation-stabilized nuclear supercoiling by direct ion oxidation. Second, as a part of the oxidation process, hydrogen peroxide will produce potentially cytotoxic free radicals close to the DNA-linked metal site, limited in extent only by the presence of chemicals capable of reducing metal ions prior to reoxidation.     

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.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Influence of different inhibitors on the activity of theRAD54 dependent step of DNA repair inSaccharomyces cerevisiae

Summary The recombinagenic pathway of DNA repair in yeast was characterized by the effect of different inhibitors on the temperature-dependent survival after-irradiation in haploid cells of the thermoconditional mutantrad54-3. Blocking protein synthesis with cycloheximide in replicating cells caused partial inhibiton of theRAD54 dependent function but some repair activity remained detectable. This indicates that-rays can induceRAD54 activity above some constitutive level of function. Inhibition of DNA replication by hydroxyurea efficiently blocked theRAD54 dependent function in stationary-phase cells but not in logarithmic-phase cells. In logarithmic-phase cells, we found a strong inhibitory effect of caffeine on theRAD54 mediated repair process.     

Inhibition of cell cycle progression by sodium butyrate in normal rat kidney fibroblasts is altered by expression of the adenovirus 5 early 1A gene

The effect of sodium butyrate (NaBut) on cell growth was studied in normal rat kidney (NRK) fibroblasts, and in NRK cells stably transfected with either the adenoviral gene E1A (wild-type), or mutated E1A (E1A^mut; with a deletion in the CR1 domain), or with the transforming Ha-ras (EJ) gene. The growth of all these cell lines was inhibited by milimolar concentrations of sodium butyrate (NaBut). However, whereas the NRK cells as well as the NRK-E1A^mut and NRK-ras cells were arrested in the G1 phase of the cell cycle, the NRK-E1A cells progressively accumulated in the G2 phase, suggesting that the E1A gene expression caused a leaky inhibition of G1 phase progression. The expression of late cell cycle-related genes cdc2 and PCNA (proliferating cell nuclear antigen) was not affected by NaBut in the NRK-E1A cells while it was totally suppressed in the other NRK-derived cell lines.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

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.     

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures

Summary By using mercury(II)-chloride (HgCl₂) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl₂ and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10^–5 and 2.0 x 10^–6 for HgCl₂ and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5–40 g/ml) that had no observable effect on the survival of the mutagenized cultures.     

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.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

The proton pump,high pH channels,and excitation: voltage clamp studies of intact and perfused cells ofNitellopsis obtusa

Summary The current-voltage (I/V) and conductance-voltage (G/ V) characteristics were recorded for intact and perfused (tonoplast-free) cells ofNitellopsis obtusa. In the pH₀ range 5 to 8, the I/V profile was sigmoidal and the G/V profile exhibited a maximum — these characteristics are attributed to the proton pump at the plasmalemma. The pH₀ dependence in this range was very similar to that found inChara corallina. At very alkaline pH₀ (11.0) the high conductance due to H⁺/OH^– channels was observed in intact cells but not in perfused cells. Young plants ofNitellopsis did not display bands of calcification, but did exhibit pH banding patterns in petri dishes. The pH bands were less than 5mm wide. The excitation transients in intact cells featured two peaks near the excitation threshold, but more peaks could be observed in the p.d. (potential difference) range –90 to –60 mV. The amplitude of the transients was strongly inhibited at pH₀ 11.0. In the perfused cells the currents lacked complete inhibition at some p.d. levels, but still exhibited one or two peaks. At high pH₀ this lack of inactivation was accentuated. The addition of 5 mM TEA to the outside medium abolished the excitation transients in perfused cells.Abbreviations EGTA ethylene glycol-bis (-amino-ethylether)-N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethane sulfonic acid) - TEA tetraethyl ammonium     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.