gamma-Ray mutagenesis in bacteriophage T4 is not enhanced by oxygen.

As in the induction of r mutants in bacteriophage T4 by gamma-rays, the radiation-induced reversion of T4 amber mutants to wild-type was found to depend on the product of the DNA-repair gene x of the phage. Neither the efficiency of induction of r mutants nor the efficiency of reversion of ambers was enhanced by the presence of oxygen during irradiation. T4 differed in this respect from phage T7, for which no indication has been found that gamma-ray mutagenesis results from error-prone repair of DNA damage. Notwithstanding the substantial contribution of misrepair to mutation induction in T4, the efficiency of induction per base-pair observed for irradiation under oxygen was lower than that found previously for T7.     

Suppression of gamma ray-induced illegitimate recombination in Escherichia coli by the DNA-binding protein H-NS

To study the mechanism of gamma-ray-induced illegitimate recombination, we examined the formation of lambdabio transducing phage in Escherichia coli after gamma-ray irradiation. We show that gamma-ray irradiation enhances the formation of lambdabio transducing phage during prophage induction. Moreover, an hns mutation synergistically enhanced the incidence of lambda-ray-induced illegitimate recombination. Next we determined the sequences at the recombination junctions of the lambdabio transducing phages induced by gamma-ray irradiation. Most of the recombination sites coincided with known hotspots. Among them, hotspot I accounted for 67% and 77% of gamma-ray-induced lambdabio transducing phages in the wild type and the hns mutant, respectively. Therefore, the recombination sites appear to occur mostly at hotspot I or at other hotspots, but rarely at non-hotspot sites. These results suggest that types of DNA damage other than the double-strand breaks induced at random sites are mainly responsible for the introduction of the site-specific or region-specific DNA double strand breaks that lead to recombination at the hotspots. The results also showed that the recombination events took place between DNA sequences possessing short stretches of homology. H-NS protein, which binds to curved DNA, suppresses illegitimate recombination in the presence and absence of gamma-ray irradiation. Models for gamma-ray-induced illegitimate recombination are discussed.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.     

Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.     

Autoradiographic Study of Gamma-Ray Induced Unscheduled DNA Synthesis in Bean Root Meristem Cells

The gamma-ray induced unscheduled DNA synthesis in root meristem cells of Vica faba was studied autoradiographically by calculating the number of cells with different 3H-thymidine labelling degree. It was found that the level of unscheduled synthesis in cells with intermediate dose (500 R) irradiation was higher than that in cells with lower dose (250 R) irradiation; however, higher dose (1000 R) irradiation would inhibit the reparative replication.     

Gamma-ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines

Well characterized gamma-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Normal human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline sucrose gradient centrifugation, repression of DNA synthesis by low doses of gamma-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression in synthesis of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW DNA synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.     

Genetic dissimilarity of putative gamma-ray-induced 'Preciosa-AAAB-Pome type' banana (Musa sp) mutants based on multivariate statistical analysis

Bananas are among the most important fruit crops worldwide, being cultivated in more than 120 countries, mainly by small-scale producers. However, short-stature high-yielding bananas presenting good agronomic characteristics are hard to find. Consequently, wind continues to damage a great number of plantations each year, leading to lodging of plants and bunch loss. Development of new cultivars through conventional genetic breeding methods is hindered by female sterility and the low number of seeds. Mutation induction seems to have great potential for the development of new cultivars. We evaluated genetic dissimilarity among putative 'Preciosa' banana mutants generated by gamma-ray irradiation, using morphoagronomic characteristics and ISSR markers. The genetic distances between the putative 'Preciosa' mutants varied from 0.21 to 0.66, with a cophenetic correlation coefficient of 0.8064. We found good variability after irradiation of 'Preciosa' bananas; this procedure could be useful for banana breeding programs aimed at developing short-stature varieties with good agronomic characteristics.     

Nm23-H1 is responsible for SUMO-2-involved DNA synthesis induction after X-ray irradiation in human cells

Human cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis activity after X-ray irradiation which is suggested to be casually related to reduction in cellular amounts of small ubiquitin-like protein modifier (SUMO-2/SMT-3A). In the present study, an increased level of DNA synthesis activity was found 8 h after X-ray irradiation in HeLa cells with reduction in SUMO-2 amounts by siRNA treatment for SUMO-2. When comparative proteomic analysis was performed between the siRNA and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, three proteins were identified as candidates. Our research focused on Nm23-H1, a nucleoside diphosphate kinase, whose amounts decreased after X-ray irradiation in HeLa cells treated with siRNA for SUMO-2. In the Nm23-H1 siRNA treated cells, induction of DNA synthesis was also detected. Furthermore, in synchronized HeLa cells, DNA synthesis was confirmed in the S phase. Moreover, increased expression of proliferating cell nuclear antigen (PCNA) was observed in Nm23-H1 siRNA treated HeLa cells after X-ray irradiation. In addition, Nm23-H1 was modified with SUMO-2 after X-ray irradiation. The present findings suggest that the reduction of Nm23-H1 is related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis via the regulation of PCNA expression after X-ray irradiation.     

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.     

Ultraviolet induction of prophage lambda during inhibition of deoxyribonucleic acid synthesis by hydroxyurea.

Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) precursors and causes the cessation of DNA synthesis. It did not cause induction of lambda. Superinfection of an irradiated lysogen with lambdaind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of lambda prophage to occur.     

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.     

The effect of the anoxic radiosensitizing agent TAN on induction of revertants by gamma-rays and helium ions in Salmonella tester strains.

The modification effect of the anoxic radiosensitizer TAN on the mutagenesis in various Salmonella tester strains after gamma-ray and helium ion irradiation was studied. The oxygen enhancement ratios (OER) for all 3 strains on the lethal assay after gamma-irradiation are approximately equal to 2. The induction of reversions in TA98 and TA100 does not modify under anoxia. The value of OER on the mutagenic assay in TA102 equals 1.6. The OER after helium ion irradiation on the lethal and mutagenic assays was less than after gamma-irradiation. The mutagenesis in 3 strains after irradiation under anoxia is enhanced by TAN. The value of the TAN modification effect after gamma-irradiation increases from 2.1 +/- 0.2 for TA102 to 5.2 +/- 0.4 for TA100. However, the TAN influence on mutagenesis in TA100 after helium ion irradiation decreases to 3.1 +/- 0.3. We conclude that peculiarities of mutagenesis in various tester strains under anoxia with TAN can be explained by considering the nature of premutational DNA damages.     

Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression.

Summary The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or -rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976). Synthesis of large quantities of protein X is induced by UV, -rays, MMC treatment or tif expression in rec ⁺ but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA ⁺ cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation.Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage or septation.     

Properties of Haemophilus influenzae mutants that are slightly recombination deficient and carry a mutation in the rec-1 gene region.

The highly recombination-deficient rec-1 mutants of Haemophilus influenzae are, as far as tested, equivalent to recA mutants of Escherichia coli. By selection for mutations in the rec-1 gene of H. influenzae, mutants designated ird (intermediary recombination-deficient) mutants were isolated; these mutants were much less recombination deficient (degree of transformability, 0.2 to 30% of wild-type value) than previously isolated rec-1 mutants (degree of transformability, 0.0001% of wild-type value). The ird mutants were more sensitive to ultraviolet irradiation and mytomycin C treatment than the wild type, but less sensitive than rec-1 mutants. Spontaneous production of phage HP1c1 by lysogenic MC11 cells and prophage induction by mitomycin C or ultraviolet irradiation were the same as in the wild type. In the ird mutants endogenous deoxyribonucleic acid was degraded both spontaneously and after ultraviolet irradiation to the same extent as in the wild type. Examination of one of the ird mutants revealed that recombination could be enhanced by ultraviolet irradiation, possibly because of an increased synthesis of the rec-1 gene product induced by ultraviolet irradiation.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

The thyroid hormone receptor-alpha (TRalpha) gene encoding TRalpha1 controls deoxyribonucleic acid damage-induced tissue repair

The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-alpha1 (TRalpha1), intestinal crypt cell proliferation in the mouse. In order to understand whether this receptor also plays a role in intestinal regeneration after DNA damage, we applied a protocol of gamma-ray irradiation and monitored cell proliferation and apoptosis at several time points. In wild-type mice, the dose of 8 Gy induced cell cycle arrest and apoptosis in intestinal crypts a few hours after irradiation. This phenomenon reverted 48 h after irradiation. TRalpha(0/0) mutant mice displayed a constant low level of proliferating cells and a high apoptosis rate during the period of study. At the molecular level, in TRalpha(0/0) animals we observed a delay in the p53 phosphorylation induced by DNA damage. In our search for the expression of the protein kinases responsible for p53 phosphorylation upon irradiation, we have focused on DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The number of cells expressing DNA-PKcs in crypts remained high 48 h after irradiation, specifically in TRalpha mutants. Altogether, in TRalpha(0/0) animals the rate of apoptosis in crypt cells remained high, apparently due to an elevated number of cells still presenting DNA damage. In conclusion, the TRalpha gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.     

Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light.

Normal human fibroblasts synthesized heat shock protein (HSP) 72 constitutively and its expression was augmented 6 hours after UV irradiation. Maximum induction of HSP72 was obtained at 12 hours and HSP72 showed a punctuated distribution in nucleus. While unscheduled DNA synthesis was almost completed 12 hours after UV irradiation, the S phase fraction decreased immediately and recovered after 6 hours. Thus, HSP72 augmentation was occurred coincidentally with the recovery of S phase, and suggested that HSP72 had some function during the recovery of DNA replication inhibited after UV irradiation.