Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells

We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T). The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells. This permeabilization can be reversed by diluting the cells in growth medium. Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation. Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells. A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells. Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type. It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion. It has a molecular weight (MW) in the range 20-30 000 D. The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible.     

Effect of cycloheximide on pools of deoxyribonucleoside triphosphates

Inhibition of protein synthesis by cycloheximide blocks DNA replication in many eukaryotic cells. To test whether this effect was mediated through enzymes furnishing DNA precursors, pool sizes of deoxyribonucleoside triphosphates were measured following cycloheximide treatment in the synchronous mitotic cycle of Physarum. It was found that cycloheximide either did not affect the pool size of DNA precursors (dATP and dGTP) or it led to a pool expansion (dCTP and dTTP). It is concluded that the arrest of DNA replication by inhibitors of protein synthesis is not due to a lack of precursors.     

Radioresistant DNA synthesis: an intrinsic feature of ataxia telangiectasia

DNA synthesis in 6 ataxia langiectasia (AT) cell strains was much more resistant to X-irradiation than was DNA synthesis in normal human diploid cells. 3 of the cell strains tested have been classified as proficient in repair replication. These data, along with those reported elsewhere, strongly suggest that radioresistant DNA synthesis is an intrinsic feature of this disease.The radioresistance of DNA synthesis in AT cells is primarily due to a reduced inhibition of replicon initiation compared to that occuring in normal cells, but DNA chain elongation is also more radioresistant in AT cells. The small inhibition of DNA synthesis that does occur in AT cells at doses up to 2000 rad is almost exclusively due to inhibition of replicon initiation and not to inhibition of chain elongation, as would be expected from results with normal human cells or from previous studies with established cell lines.     

DNA synthesis in polyoma virus infection. III. Mechanism of inhibition of viral DNA replication by cycloheximide.

The formation of viral DNA was inhibited in polyoma virus-infected cells in which protein synthesis had been blocked by cycloheximide. The present studies show the following. (i) The pool of replicating viral DNA molecules was reduced in cycloheximide-treated cells by an amount consistent with inhibition of [3-H]thymidine incorporation into viral DNA, whereas the rate of turnover of the replicating population was not affected. (ii) The rate of conversion of replicating molecules into closed-circular DNA was not affected by cycloheximide. (iii) The rate of elongation of nascent viral DNA fragments into strands of unit genome length was unaffected by cycloheximide. It is concluded that viral DNA synthesis is inhibited in the absence of protein synthesis exclusively at the level of initiation of new rounds of genome replication. Replicating molecules already initiated at the time of addition of cycloheximide matured into progeny closed-circular DNA at a normal rate.     

Analysis of Simian Virus 40-Induced Transformation of Hamster Kidney Tissue in Vitro VIII. Induction of Infectious Simian Virus 40 from Virogenic Transformed Hamster Cells by Amino Acid Deprivation or Cycloheximide Treatment

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.     

3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells

3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADP-ribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.     

Tn10 transposition does not respond to environmental stress

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Inducible processes in DNA replication and repair after gamma, UV-irradiation and action of some chemicals in mammalian cells

Restored DNA synthesis in mammalian gamma-, UV-irradiation and action of FdUrd was shown to be resistant to gamma- and UV-irradiation or heating. This correlates well with changes in chromatin structure and perhaps depends on the modification of the latter. For studying possible inducible characteristics of restored process of DNA synthesis the irradiated cells were incubated with cycloheximide (1 or 10 micrograms ml-1) or actinomycin D (0.05 ug ml-1). It was shown that in the presence of cycloheximide or actinomycin D restoration of DNA synthesis did not occur. A high rate of postreplicative DNA repair in UV-irradiated HeLa cells occurs after the previous action of FdUrd or UV-irradiation. Under these conditions daughter DNA strands have few gaps. Two-dimensional gel electrophoresis of proteins from the cells with resistant DNA synthesis demonstrates higher level some of these and lower one of the other proteins.     

DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication.

Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.     

Sister chromatid exchanges, during x-irradiation, in the blood lymphocytes of patients with hereditary diseases and radioresistant DNA synthesis

X-ray irradiation induced sister chromatid exchanges (SCE) in blood lymphocytes from patient with Down's syndrome and adult progeria (in both the cases radioresistant DNA synthesis takes place). In these diseases, likely as upon form II of xeroderma pigmentosum (the replicative DNA synthesis is radioresistant), X-ray irradiation lowers the rate of SCE compared with that in the control, then the SCE rate rises with the increase in radiation dose, reaching the rate of SCE in non-irradiated cells. In normal lymphocytes (in which ionizing radiation inhibits the replicative synthesis of DNA) the rate of SCE rises with the rise of radiation dose. Thus, the rate of SCE in X-ray irradiated lymphocytes is in reverse dependence with radioresistance of replicative synthesis of DNA. The data obtained are explained in accordance with the replicative hypothesis of the SCE nature (Painter, 1980a): in cells of patients with Down's syndrome, xeroderma pigmentosum form II and progeria of adults the time of existence of partly replicated clusters of replicons is decreased due to radioresistant replicative synthesis of DNA, but the presence of partly replicated clusters of replicons is necessary for SCE formation. Therefore the rate of SCE in X-irradiated cells of these patients decreases.     

Regulation of the G1 leads to S phase transition in chick embryo fibroblasts with alpha-keto acids and L-alanine.

Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum-stimulated DNA synthesis in G1-arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H-thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4 degrees C while exposed to the drug. Several alpha-keto acids (pyruvate, oxaloacetate, alpha-ketobutyrate) at 0.5-1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L-alanine (D-alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Glucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin-stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for alpha-keto acid or L-alanine. A serum component(s) with a molecular weight of about 100 permitted insulin-stimulated DNA synthesis in inhibited cells.     

DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns.

A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.     

DNA replication in mammalian cells after exposure to factors of a physical, chemical or biological nature. III. 5-Fluorodeoxyuridine induces DNA synthesis in cells not suppressed by gamma irradiation

The influence of preincubation of HeLa and Chinese hamster V79 cells with fluorodeoxyuridine (FUdR, 10(-6) M) on DNA replication and molecular weight of nascent DNA was studied after gamma-irradiation with a dose as much as 10 Gy. The 60Co-radiation inhibits DNA synthesis in both HeLa and V79 cells by 30-40 per cent. The incubation with FUdR before irradiation suppresses the inhibitory effect of irradiation on DNA synthesis. It is suggested that differences in gamma-radiation inhibition of DNA synthesis may result from the FUdR-induced changes in chromatin structure, rather than from synchronization of cell growth. This suggestion is based on the observation that the radioresistant mode of DNA synthesis occurred 18 hours following the short-term (6 hours) incubation with FUdR in cell cultures differing from each other in almost 2-fold their cell longevity.     

Coupling of histone mRNA levels to radioresistant DNA synthesis in ataxia-telangiectasia cells

Cloned genomic DNA for human histone H1, H3 and H4 genes has been used to determine the effects of -radiation on histone mRNA levels and synthesis in ataxia-telangiectasia cells. Synthesis of histone mRNA was determined in cells synchronized with aphidicolin. Effects of irradiation on DNA synthesis and passage through S phase were also monitored. Irradiation was found to slow the passage of control cells through the cell cycle but had no effect on progression of ataxia-telangiectasia cells. H1 and core histone mRNA synthesis was inhibited by radiation in two control cell lines after release from aphidicolin block. No inhibition was observed in one ataxia-telangiectasia cell line and a small degree of inhibition in a second. An increased level of mRNA was observed in both irradiated control and ataxia-telangiectasia cells at 5–7 h post-irradiation compared to unirradiated cells. Similar results were obtained in log phase cells. These results demonstrate that histone mRNA synthesis is radioresistant in ataxia-telangiectasia cells and is coupled to radioresistant DNA synthesis in these cells.     

Survival, Deoxyribonucleic Acid Breakdown, and Synthesis in Salmonella typhimurium as Compared with Escherichia coli B Strains

Salmonella typhimurium LT-2 was compared with radioresistant (B/r) and radiosensitive (B(s-2)) strains of Escherichia coli in respect to the survival, deoxyribonucleic acid (DNA) breakdown, and DNA synthesis after X irradiation. It is shown that S. typhimurium LT-2 is about four times more sensitive than E. coli B/r but less sensitive than B(s-2). The DNA breakdown is in S. typhimurium LT-2 lower than the postirradiation breakdown of DNA in both E. coli strains and DNA synthesis proceeds in this bacterium in spite of a much lower survival, as in the radioresistant E. coli B/r.     

Inhibition of DNA synthesis in senescent-proliferating human cybrids is mediated by endogenous proteins

Cytoplasts were prepared from senescent human diploid fibroblasts. Brief treatments of the senescent cells with cycloheximide or puromycin prior to or after enucleation eliminated the ability of senescent cytoplasts to block initiation of DNA synthesis in senescent-young cybrids. Senescent cells treated with cycloheximide, enucleated and allowed to recover in complete medium without cycloheximide, regained the ability to block initiation of DNA synthesis in senescent-young cybrids. These results support the hypothesis that senescent cells synthesize an inhibitor of DNA synthesis which is either a protein(s) or its activity is mediated by a protein(s) found in the cytoplasm of the senescent cell.     

The relationship between the radiosensitivity of DNA replicative synthesis and the formation of sister chromatid exchanges]

It has been found that irradiation in doses 0.5-2.0 Gy does not enhance the frequency of sister chromatid exchanges in cells of patients with Down's syndrome and ataxia-telangiectasia compared to the normal cells. In the case of ataxia, this phenomenon was accompanied with radioresistant replicative DNA synthesis, whereas in two cases of Down's syndrome the replicative DNA synthesis was found to be as radiosensitive as in the norm. According to these data, the mechanism of sister chromatid exchanges proposed in our previous publication (Pleskach et al., 1988) seems to be rather doubtful.     

Effect of protein synthesis inhibitors on DNA replication. I. Effect of the short-term action of cycloheximide on cell permeability for thymidine in vitro]

The effect of protein synthesis inhibitors on DNA replication was studied on L cells. After a 10 minutes' action of the inhibitors, protein synthesis was seen to be completely blocked, and DNA synthesis decreased by 85%. Four hours after a 20-minutes' cycloheximide treatment, the cells completely restored their ability to protein synthesis and DNA replication and even surpass the control level, due, probably, to a partial cell synchronization in S period. The short action of cycloheximide did not interfere with thymidine uptake by the cells. The rate of the exogenous precursor uptake was even higher than that in the control, apparently, because of its much reduced utilization in the inhibited DNA synthesis.     

Effects of cycloheximide on chromatin biosynthesis.

In the presence of sufficient cycloheximide, puromycin or NaCl to quantitatively inhibit protein synthesis in HeLa cells, thymidine incorporation continues at 20% of control rates for 60 to 90 minutes, after which incorporation gradually ceases. Both DNA and protein synthesis revert to control rates in about five minutes after removal of cycloheximide.DNA synthesis in the presence of cycloheximide appears to be a continuation of the replicative process by several criteria. The persistent DNA synthesis in the presence of cycloheximide is abolished by hydroxyurea, which does not inhibit repair synthesis, while ethidium bromide, an inhibitor of mitochondrial DNA synthesis, is without effect. Nuclear DNA is not nicked during incubation in cycloheximide. Low molecular weight Okazaki fragments (4 to 5 S) are both synthesized and processed to high molecular weight DNA in cells treated with cycloheximide. Replication forks, identified in alkaline CsCl gradients by incorporation of bromodeoxyuridine as a density marker just before the addition of cycloheximide, are selectively labeled with radioactive thymidine during DNA synthesis.In the presence of cycloheximide the maturation of DNA intermediates into high molecular weight DNA is defective. All size classes of DNA fragments, normally present during progression of low to high molecular weight DNA, are demonstrable in cells preincubated in cycloheximide for prolonged periods. However, 21 S fragments, intermediate in size between Okazaki pieces and mature, high molecular weight DNA, accumulate in cells treated with cycloheximide, demonstrating a defect in maturation of the 21 S intermediates into high molecular weight DNA. After removal of the cycloheximide, the 21 S DNA fragments are processed to high molecular weight DNA at a significantly impaired rate, requiring about three hours for completion of chain growth as compared to 40 to 60 minutes in controls. The slowed growth of DNA fragments synthesized in the presence of cycloheximide following drug removal is not due to persisting effects of cyeloheximide since DNA synthesis immediately following removal of the drug has chain growth rates similar to that of controls.Pools of chromatin proteins exist in HeLa cells, as demonstrated by a brief, labeled amino acid pulse followed by a chase with cycloheximide. The specific activity of chromatin proteins increases significantly during 60 minutes of cycloheximide inhibition. Histone f2a1 accumulates preferentially during this chase period, suggesting that a supply of this highly conserved histone might be requisite to continued replication.Comparison of chromatin synthesized during cycloheximide treatment with pulse-labeled control chromatin has provided insight into the mechanism of assembly of proteins and DNA into the nucleoprotein complex. The DNA of ch-chromatin² is more susceptible to nuclease digestion than control chromatin, suggesting that it is deficient in protein content. Upon reversal of cycloheximide inhibition, the recovery of nuclease digestibility of ch-chromatin to control values takes two to three hours, a time similar to that required for conversion of the corresponding 21 S chDNA fragments to high molecular weight DNA. Briefly pulse-labeled (30 to 60 s) DNA in control chromatin also has an enhanced susceptibility to nuclease digestion of the same degree as found in ch-ehromatin. The time of recovery of increased nuclease susceptibility of newly made chromatin DNA (via protein addition) to control levels is about 10 to 15 minutes and corresponds to the time required for synthesis of replicon-sized units of DNA.In addition to being nuclease-sensitive, both cycloheximide and newly synthesized (30 to 60 s) chromatin have lighter buoyant densities in CsCl gradients than bulk chromatin. This property exists for only one to two minutes in controls and is probably due to structural properties distinct from those rendering nuclease sensitivity.Limit digests of chromatin by micrococcal nuclease yield a characteristic pattern of polynucleotides when resolved in polyacrylamide gels. The radioactivity profiles of limit digest polynucleotides from control and ch-chromatin are identical, indicating that pre-existing chromatin proteins remain in place on newly replicated DNA in the same fashion as in mature chromatin.     

Gamma-irradiation induces in HeLa cells radioresistant DNA synthesis

Cells of a suspension HeLa culture at the logarithmic phase of growth were exposed to 60Co-gamma-rays (5 Gy), incubated in the nutritious medium, and in 4 h subjected to repeated irradiation: the dose-response function and the dynamics of DNA synthesis inhibition were determined. It was shown that DNA synthesis was inhibited to a lesser extent after preirradiation, in other words, DNA synthesis was radioresistant. A correlation between this synthesis and reproductive cell death is discussed.