The effect of repeated small doses of radiation on recovery from sub-lethal damage by Chinese hamster cells irradiated in the plateau phase of growth.

Unfed plateau-phase cells have been irradiated with either single doses or up to ten fractions of X-rays 6 hours apart. The single-dose survival curve had an extrapolation number of 11-4, and the oxygen-enhancement ratio (o.e.r.) was 3-1. Cells were exposed to multiple fractions of 200 rad or 150 rad in air and 600 rad or 450 rad in hypoxia. The resulting survival curves did not fit a multi-target, single-hit model of cell survival, being much steeper than that would predict. The curves were exponential up to five fractions of X-rays, but tended to bend downwards with increasing number of fractions. Cells that had survived five fractions of 200 rad (or 600 rad in hypoxia) 6 hours apart, were less able to absorb damage as sub-lethal than those which had not previously been exposed to radiation. The ratio of the initial slopes of the fractionated survival curves for irradiation in air and hypoxia was 2-1, implying that the o.e.r. "on the shoulder" may be less than that in the exponential region of survival.     

Effects of tritiated water on germ cells in medaka embryos

Embryos of medaka, Oryzias latipes, were exposed to tritiated water and 137Cs gamma rays continuously from the one-cell stage until hatching (10 days at 26 degrees C). Germ cells in the gonads of newly hatched fry were counted in histological sections and compared with controls. The accumulated dose for 50% survival of germ cells was 195 rad for tritium beta rays and 350 rad for 137Cs gamma rays. Female progeny were produced using Yamamoto's method. The 50% survival doses for female germ cells treated in a manner similar to that described above were 140 rad for beta rays and 305 rad for gamma rays. When embryos of medaka were irradiated with gamma rays below an accumulated dose of 475 rad or treated with tritiated water at a concentration of 0.2 mCi/ml or lower, the dose response of the germ cells showed an exponential relationship. It appeared that there was no threshold or significant dose-rate effect for either beta or gamma rays on germ cell survival, and that tritium beta rays were more effective than 137Cs gamma rays in germ cell killing.     

Roles of <Emphasis Type="Italic">Saccharomyces cerevisiae RAD17</Emphasis> and <Emphasis Type="Italic">CHK1</Emphasis> checkpoint genes in the repair of double-strand breaks in cycling cells

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background. Nelson Bracesco and Ema C. Candreva have contributed equally to this article.     

Ozone response in wild type and radiation-sensitive mutants of Saccharomyces cerevisiae.

Summary The effect of ozone exposure on Saccharomyces cerevisiae was studied. Factors such as ozone concentration, treatment time, media, initial cell concentration and growth phase were shown to influence ozone response in this organism. Logarithmic phase cells were much more sensitive than stationary phase cells to the lethal effect of ozone.The radiation-sensitive mutants rad3, rad6, rad51 and rad52 of S. cerevisiae were exposed, in water, to 50 ppm of ozone for 30 min. On comparing their survival curves, the rad51 and the rad52 mutants showed a greater sensitivity to ozone exposure than the wild type.     

Repair of gamma-ray induced DNA strand breaks in the radiation-sensitive mutant rad18-2 of Saccharomyces cerevisiae

The repair of gamma-ray induced DNA single and double-strand breaks was looked at in wild type and rad18-2 strains of the yeast Saccharomyces cerevisiae using sucrose gradient centrifugation. It was found that rad18-2 diploid cells could repair single and double-strand breaks induced by gamma-rays. It was also found that rad18-2 cells experienced a breakup of their DNA during post-irradiation incubation to a size smaller than seen in cells just receiving irradiation. This breakup of DNA in rad18-2 cells is not degradation due to cell death since wild type cells irradiated to similar low survival levels do not show this breakup of DNA with 8 h incubation. The breakup of DNA in rad18-2 cells is not due to replication gaps being formed by synthesis on a damaged template since treatment of rad18-2 a mating type cells with alpha factor, to prevent initiation of DNA synthesis, does not prevent breakup of the DNA.     

The checkpoint protein Rad24 of Saccharomyces cerevisiae is involved in processing double-strand break ends and in recombination partner choice

Upon chromosomal damage, cells activate a checkpoint response that includes cell cycle arrest and a stimulation of DNA repair. The checkpoint protein Rad24 is key to the survival of a single, repairable double-strand break (DSB). However, the low survival of rad24 cells is not due to their inability to arrest cell cycle progression. In rad24 mutants, processing of the broken ends is delayed and protracted, resulting in extended kinetics of DSB repair and in cell death. The limited resection of rad24 mutants also affects recombination partner choice by a mechanism dependent on the length of the interacting homologous donor sequences. Unexpectedly, rad24 cells with a DSB eventually accumulate and die at the G(2)/M phase of the cell cycle. This arrest depends on the spindle checkpoint protein Mad2.     

8-Methoxypsoralen plus 365 nm light effects and repair in yeast.

Haploid wild-type and mutant cells of Saccharomyces carrying one of the single genes rad2-20 or rad9-4 and the double mutant rad2-20rad9-4 were tested for their response to a treatment with 8-methoxypsoralen plus 365 nm light using immediate and delayed plating techniques. The mutant defective in the excision of ultraviolet-induced pyrimidine dimers (rad2-20) as well as that presumably deficient in a recombinational repair system (rad9-4) are more sensitive than wild type cells. The double mutant (rad2-20rad9-4) demonstrates a higher sensitivity than each of the single mutants, indicating that at least two pathways are involved in the repair of the 8-methoxypsoralen plus 365 nm induced damages. In all cases survival curves have shoulders. The survival of wild type and rad9-4 cells is increased after dark holding whereas it remains constant for the rad2-20 mutant and for the double mutant. These results show that the induced damages are reparable. Respiratory deficient mutant (p-) were compared to the corresponding respiratory competent cells. It is shown that the respiratory function is required for the expression of the excision repair activity. The 8-methoxypsoralen plus 365 nm ligh treatment appears to be less effective than ultraviolet irradiation (254 nm) in the induction of the cytoplasmic 'petite' mutation at the same survival levels.     

Reparation after the action of 8-methoxypsoralen and light (lambda=365 nm) on radiosensitive mutants of Saccharomyces cerevisiae yeasts]

The method of repeated irradiation allowed to study kinetics of excision of mono-adducts induced by 8-methoxypsoralen (8-MOP) plus light (lambda=365 nm) in DNA of UV-sensitive mutants rad4 and rad15 and X-ray sensitive mutants rad54, xrs2, xrs4. The survival of the mutant rad4 was not practically increased after incubation in complete liquid medium for 3 hours at 28 degrees C before the repeated irradiation. These data suggest that the mutant rad4 is characterized by nearly complete absence of the mono-adduct excision. The survival of mutants rad15 and rad54 in the same environment was increased less effectively than the survival of the control radioresistant strain, but the mutants xrs2 and xrs4 did not differ from the control strain. Possible causes of differences in survival between radiosensitive strains are discussed. The increased sensitivity of the excision defective strain (rad4) and of the postreplicative recombination defective strains (xrs2, xrs4, rad54) to the lethal effect of 8-MOP plus light (lambda=365 nm) suggests that two systems of reparation take part in the removal of photoproducts induced by 8-MOP in DNA of yeast cells.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

Strain differences in the response of mouse testicular stem cells to fractionated radiation

The survival of spermatogonial stem cells in CBA and C3H mice after single and split-dose (24-hr interval) irradiation with fission neutrons and gamma rays was compared. The first doses of the fractionated regimes were either 150 rad (neutrons) or 600 rad (gamma). For both strains the neutron survival curves were exponential. The D0 value of stem cells in CBA decreased from 83 to 25 rad upon fractionation; that of C3H stem cells decreased only from 54 to 36 rad. The survival curves for gamma irradiation, which all showed shoulders, indicated that C3H stem cells had larger repair capacities than CBA stem cells. However, the most striking difference between the two strains in response to gamma radiation was in the slopes of the second-dose curves. Whereas C3H stem cells showed a small increase of the D0 upon fractionation (from 196 to 218 rad), CBA stem cells showed a marked decrease (from 243 to 148 rad). The decreases in D0 upon fractionation, observed in both strains with neutron irradiation and also with gamma irradiation in CBA, are most likely the result of recruitment or progression of radioresistant survivors to a more sensitive state of proliferation or cell cycle phase. It may be that the surviving stem cells in C3H mice are recruited less rapidly and synchronously into active cycle than in CBA mice. Thus, it appears that the strain differences may be quantitative, rather than qualitative.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Survival of diploid yeasts after gamma irradiation: genetic control of the maturation effect

A study was made of the influence of rad mutation, leading to radiosensitivity increase, on the effect of additional growth in diploid Saccharomyces cerevisiae yeast exposed to gamma-radiation. The most radiosensitive mutants of this series, rad52/rad52 and rad54/rad54, did not virtually vary from the wild type cells in the value of the additional growth effect. Some other mutants, for instance, rad53/rad53 and rad55/rad55, exhibited a significantly lesser effect of additional growth. It was shown that the effect of additional growth did not depend upon the rate of rapid and slow postirradiation recovery of the wild type cells. The results of the studies prompt the conclusion that the processes responsible for the additional growth effect and those responsible for recovery of cells from radiation damages are mutually independent.     

Variation in U.V. primary fluorescence-intensity of vital cells depending on 60Co gamma-radiation dose.

Using impulse-cytofluorophotometry in the ultra-violet spectral region the author has shown on vital, unstained Ehrlich ascites tumour cells that the primary fluorescence intensity of this tumour is on day 11 after transplantation 20 per cent higher than on day 8. Storage of the vital cells for 25 min at 20 degrees C has no effect on this result. When the cells are exposed to 60Co to gamma-radiation on day 6, a new stable fluorescence level is established afted 20 hours. Measurements of the primary fluorescence intensity depending on dose have shown a significant rise starting from 75 rad at 48 hours after irradiation. The fluorescence intensity rises by 42.5 per cent of the control value at 3000 rad, but only by 31.5 per cent on exposure to 4000 rad.     

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52-3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.     

The in vitro radiosensitivity of hemopoietic stem cells from control and preirradiated infant mice

Summary The radiosensitivity of hemopoietic stem cells isolated from infant mice (6 or 9 days of life), of infant preirradiated mice (exposed to 126 rad on day 6 and assayed at day 9 of life) and of adult C57/B1 mice was assayed on the basis of their capacity to form spleen colonies and to incorporate iododeoxyuridine after transplantation into heavily irradiated hosts. Stem cells of infant non-irradiated mice have a D₀ of 115 rad compared to 72 rad for adult mice whereas the D₀ of preirradiated infant mice has diminished to 80 rad. No significant difference in D₀ was seen between spleen and bone marrow cells or between total cells and cells not sensitive to³H-thymidine. It is postulated that this sensitization of stem cells caused by a preirradiation is responsible for the greater mortality of infant mice after fractionated exposure compared to a single one.     

Inducible error-prone repair in yeast. Suppression by heat shock

The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same treatment after mutagen exposure did not lower the induced mutation frequency. In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure. These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors. The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents. A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation. Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light. These results indicate that, unlike the SOS system of E. coli, this inducible error-prone process of yeast is responsive to only certain mutagens. Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18). The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques. These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.     

Kinetics of uptake, retention, and radiotoxicity of 125IUdR in mammalian cells: implications of localized energy deposition by Auger processes

The kinetics of uptake, retention, and radiotoxicity of 125IUdR have been studied in proliferating mammalian cells in culture. The radioactivity incorporated into the DNA is directly proportional to the duration of incubation and to the extracellular concentration of 125I. The rate of proliferation of cells is related to the intracellular radioactive concentration and is markedly reduced at medium concentrations greater than or equal to 0.1 mu Ci/ml. At 37% survival the high LET type cell survival curve is characterized by an uptake of 0.035 pCi/cell, and the cumulated mean lethal dose to the cell nucleus is about 80 rad compared to 580 rad of X-ray dose for this cell line. The strong cytocidal effects of the decay of 125I correlate with localized irradiation of the DNA by the low energy Auger electrons.     

Saccharomyces cerevisiae rad51 mutants are defective in DNA damage-associated sister chromatid exchanges but exhibit increased rates of homology-directed translocations.

Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Delta3'::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Delta3'::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.     

Cisplatin DNA cross-links do not inhibit S-phase and cause only a G2/M arrest in Saccharomyces cerevisiae.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.