DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae.

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

Phototherapeutic, photobiologic, and photosensitizing properties of khellin.

Khellin, whose chemical structure closely resembles that of psoralen, is reported to be an efficient drug for treating vitiligo when combined with ultraviolet A irradiation. Photobiological activity on yeast is found to be much lower than that of bifunctional psoralens such as 5-methoxypsoralen. In vitro experiments reveal that khellin is a poor photosensitizer. It behaves as a monofunctional agent with respect to DNA photoaddition. It does not photoinduce cross-links in DNA in vitro or in Chinese hamster cells in vivo. This behavior may explain the low photogenotoxicity in yeast and the lack of phototoxic erythemal response when treating vitiligo with khellin.

Detection of pyrimidine dimers and monoadducts induced by 7-methylpyrido(3,4-c) psoralen and UVA in Chinese hamster V79 cells by enzymatic cleavage and high-pressure liquid chromatography.

The photochemotherapeutically active psoralen derivative 7-methylpyrido(3,4-c) psoralen (MePyPs) has been recently shown to be able to photoinduce monoadducts of the C4-cycloaddition type as well as pyrimidine dimers in DNA in vitro. In the present study, we report on the induction of these two types of photolesions in mammalian cells in culture. The MePyPs photocycloadducts were quantified in V79 Chinese hamster cells after treatment with MePyPs plus UVA following enzymatic hydrolysis of the DNA by DNase I, S1 nuclease and acidic phosphatase treatments. Concomitantly induced pyrimidine dimers were determined by two methods, high-pressure liquid chromatography and alkaline gel electrophoresis after dimer-specific endonucleolytic cleavage. The results show that, in Chinese hamster cells treated with MePyPs plus UVA, the yield of pyrimidine dimers is approximately 5-10% that of MePyPs-DNA photocycloadducts. Because psoralen monoadditions to DNA alone are generally not considered as being very phototoxic, a synergistic interaction of monoadditions with pyrimidine dimers may be expected to occur in order to explain the high photobiological effectiveness of this psoralen derivative.

Induction and removal of DNA interstrand cross-links in V-79 Chinese hamster cells measured by hydroxylapatite chromatography after treatments with bifunctional furocoumarins.

DNA interstrand crosslinks (CL) photoinduced by bifunctional furocoumarins in V-79 Chinese hamster cells were measured by alkaline denaturation and hydroxylapatite chromatography. Treatments with 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (4,5',8-TMP) and 365 nm irradiation (UVA) confer a dose-dependent linear increase in the amount of double-stranded DNA indicating the induction of CL. Determination in alkaline sucrose gradients of the molecular weight of the DNA and estimation of drug-induced strand breakage allowed quantification of the CL induced. 5-MOP was found to be slightly more effective than 8-MOP whereas 4,5',8-TMP was 9 times more effective for the induction of CL. The fate of CL during post-treatment incubation was also followed. Cells in exponential growth phase were found to be efficient in the removal of CL.

Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different gamma-ray dose-rates: a pulsed-field gel electrophoresis analysis.

We investigated the effects of gamma-ray exposures at high dose-rate (HDR, 23.2 Gy/min) and low dose-rate (LDR, 0.47 Gy/min) on survival and the induction of DNA double-strand breaks (dsb) in a diploid wild-type (D7) and the repair-deficient mutant strain rad52/rad52 of Saccharomyces cerevisiae. Analysis by pulsed-field gel electrophoresis (PFGE) using a contour homogeneous electric field apparatus revealed that, at HDR, in the range 0-400 Gy, dsb are induced as a linear function of gamma-ray dose. Liquid holding recovery in non-nutrient medium (LHR) for 48 h of wild-type cells treated at HDR, significantly increased survival and reduced the yield of dsb. Such changes did not occur in rad52/rad52 cells defective in the repair of dsb. Thus, in gamma-irradiated wild-type cells, an efficient repair of dsb is taking place during LHR. Treatments of wild-type cells at LDR resulted in higher survival and an approximately two-fold lower yield of dsb than at HDR. Such a dose-rate effect was absent in rad52/rad52 cells suggesting that, in wild-type cells during LDR exposures, significant amounts of dsb can be repaired. This repair could be very much accentuated by 48-h LHR of wild-type cells treated at LDR. The relationship observed between gamma-ray survival and dsb repair clearly indicates that increases in survival of wild-type cells, during LDR as compared with HDR exposures and after LHR, are strongly related to the repair of dsb.

Pulsed-field gel electrophoresis analysis of the repair of psoralen plus UVA induced DNA photoadducts in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, double-strand breaks (DSB) have been observed during the DNA repair of psoralen plus UVA induced lesions. In the present paper, we analyzed this repair step in some detail using pulsed-field gel electrophoresis (CHEF) to get a better understanding of this phenomenon with regard to the type of lesions induced and the repair pathways involved. The results confirm that, during post-treatment incubation of Saccharomyces cerevisiae cells, DSB are formed. Their appearance is dose-dependent and the rate of induction is comparable in large (chromosome IV) and small (chromosome III) chromosomes. The formation of DSB is evidenced by the breakage of linear chromosomes III and IV, but also, after high doses, by the linearization of a circular form of chromosome III. The induction of DSB appears to be highly dependent on the induction of interstrand cross-links since they are clearly present after treatments with 8-MOP plus 365 nm radiation (inducing monoadducts and cross-linking in DNA), but practically absent after treatment with 8-MOP plus 405 nm radiation (inducing predominantly monoadducts) at comparable levels of photoadducts. The occurrence of DSB is dependent on the RAD2 and RAD52, but not on the RAD6 gene. It is likely that the specific processing of DNA lesions involving DSB is related to the genotoxic consequences observed.

Induction of double-strand breaks in Chinese hamster ovary cells at two different dose rates of gamma-irradiation.

Using pulsed-field gel electrophoresis (PFGE) analysis we investigated the existence of a dose rate effect of gamma-irradiation on the measured presence of DNA double-strand breaks (DSB) in a repair competent (K1) and a repair deficient (mutant xrs6) Chinese hamster ovary (CHO) cell line. The fraction of DNA fragments released from cells embedded in agarose during PFGE after gamma-irradiation was taken as a measure of DSB induction. In CHO-K1 cells DSB were present at a significantly higher rate when gamma-irradiation was delivered at a high dose rate of 22 Gy/min (HDR) than at a medium dose rate of 0.45 Gy/min (MDR) at 37 degrees C. However, the same amount of DSB was found when irradiation was performed at the two dose rates at 4 degrees C. The DSB yield was also identical at both dose rates in the DSB repair deficient mutant xrs6. The results indicate that there is an apparent dose rate effect for gamma-ray induced DSB in repair competent CHO cells due to partial repair of DSB taking place during gamma-ray exposures at MDR but not at HDR. This repair of DSB was inhibited upon irradiation at 4 degrees C and in repair deficient xrs6 cells.

Non-specific incision of DNA due to the presence of 8-methoxypsoralen photoinduced interstrand cross-links in Saccharomyces cerevisiae.

The repair of DNA interstrand cross-links (CL) induced by 8-methoxypsoralen (8-MOP) plus UVA irradiation was analyzed by the alkaline step elution technique. A double-exposure protocol was used with 8-MOP, starting with exposure to monochromatic 405-nm radiation inducing only DNA monoadducts (MA), followed, after washing out of unbound 8-MOP molecules, by a second exposure to 365-nm radiation inducing varying relative amounts of CL at a constant level of total photoadducts. In the range of doses used for the second exposure, repair of CL took place; however, in the presence of increased relative amounts of CL induced non-specific incision of DNA occurred. This endonucleolytic cleavage appears to be related to the increased mutagenic and recombinogenic effects observed at increased levels of CL.

Repair of the two diastereoisomer photoadducts formed between 7-methylpyrido(3,4-c)psoralen (MePyPs) and thymidine in yeast cells: a chemical approach.

When analysing the repair of psoralen plus UVA-induced photoadducts in DNA, it must be realized that, in most cases, different isomers are formed. The monofunctional psoralen derivative 7-methylpyrido(3,4-c)psoralen (MePyPs) is known for its high antiproliferative activity at the cellular level and interesting photochemotherapeutic properties. To understand its photobiological efficiency in more detail, the induction of specific photoadducts in DNA and their repair were analysed in a eukaryotic cell system, the yeast Saccharomyces cerevisiae. After photoaddition of MePyPs, two main diastereoisomers were characterized after enzymatic hydrolysis of the DNA and analysis by high performance liquid chromatography. One diastereoisomer was more effectively repaired in yeast than the other during post-treatment incubation, suggesting that the two diastereoisomers may be recognized differently by cellular enzymatic repair systems.

Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells.

In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e. monoadducts and interstrand crosslinks. Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences. We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest. In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA. The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido[3,4-c]psoralen greater than 3-carbethoxypsoralen. The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters. It probably involves an inducible component. Chromosome breakage occurs during the repair period after PUVA treatment. It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.

Genetic effects and repair of DNA photo-adducts induced by 8-methoxypsoralen and homopsoralen (pyranocoumarin) in diploid yeast.

The relationship between DNA mono- and di-adducts and genetic effects induced by the pyranocoumarin 8,8-desmethylxanthyletine (homopsoralen) HP and 365 nm radiation (UVA) was investigated in the diploid yeast strain D7 (Saccharomyces cerevisiae) taking 8-methoxypsoralen (8-MOP) as a reference compound. The number of DNA cross-links (CLs) induced was determined using alkaline step elution analysis. The induction and removal of total photo-adducts was followed using radioactively labelled compounds. HP showed the same photobinding capacity as 8-MOP. As a function of UVA dose, it was less effective than 8-MOP for the induction of CLs and genetic effects. However, as a function of CLs induced, HP was shown to be more effective for the induction of lethal effects and mitotic recombination than 8-MOP but equally effective for the induction of mutations. The results suggest that, although CLs are recognized as genetically effective lesions, at a given number of CLs, HP induced mono-adducts efficiently contribute to the induction of lethal effects and mitotic recombination but less to the induction of mutations. Using a re-irradiation protocol, HP was brought to yield the same relative amounts of CLs at the same number of total adducts as single UVA exposures with 8-MOP. In these conditions, mutation induction and the kinetics for the removal of photo-adducts were the same for both agents indicating that not only the removal of adducts but also mutation induction are highly dependent on the relative level of CLs induced.

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.

ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence.

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1ß foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.

Repair of 8-methoxypsoralen photoinduced cross-links in yeast. Analysis by alkaline step-elution and electron microscopy.

The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D7 and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced cross-links. In accordance with previous reports, the D7 rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D7 rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D7 RAD+ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D7 rad18-1 than in D7 RAD+. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.

Disruption and functional analysis of six ORFs on chromosome IV: YDR013w, YDR014w, YDR015c, YDR018c, YDR020c, YDR021w (FAL1).

The disruption of six novel yeast genes has been realized in two genetic backgrounds. Six open reading frames (ORFs) from chromosome IV, YDR013w, YDR014w, YDR015c, YDR018c, YDR020c and YDR021w, were disrupted using the KanMX4 marker and PCR-targeting with long flanking regions homologous (LFH) to the target locus. The deletants were verified at the molecular level, using PCR and Southern analysis. Sporulation and tetrad analysis revealed that ORFs YDR013w and YDR021w (also known as FAL1) are essential genes. Microscopical observations showed that ydr013wDelta haploid cells were blocked after one or two cell cycles and presented heterogeneous bud sizes. The ydr021wDelta haploid cells gave rise to microcolonies of about 20 cells. The other four ORFs are non-essential. Basic phenotypic analysis of the non-lethal deletant strains did not reveal any significant differences in cell morphology, growth on different media and temperatures, sporulation and mating efficiency between parental and mutant strains in the FY1679 background.

Determination of a thermal equivalent of millimeter microwaves in living cells.

Recent microwave experiments have shown frequency dependent influences on the growth rate of bacteria. To determine whether microwaves are able to affect growth (or to induce lesions in cellular DNA of yeast cells), experiments were performed with millimeter microwaves at frequencies between 70 and 75 GHz. Saccharomyces cerevisiae cells were irradiated on millipore filter discs placed on agar plates in open petri dishes. A diploid strain of yeast (D5, Zimmerman), that is sensitive to genetic insult was used to study the effects of temperature and of microwave irradiation on cell survival, induction of mitotic recombination, and induction of cytoplasmic "petite" mutations. No evidence of altered survival, impaired function, or structural injury was seen at either frequency, even at power densities as high as 60 mW/cm2. Conventional heating had no deleterious effects until temperatures of specimens exceeded 50 degrees C. In addition, two haploid strains of yeast of opposite mating type were compared with respect to temperature and microwave treatment for formation of zygotes. The elevation of temperature due to the microwave treatment at 60 mW/cm2 and 2 mm distance was estimated to correspond to 3 degrees C.

[The effect of electromagnetic radiation of wavelength in the millimeter range on bacterial growth]. / Action d'un rayonnement électromagnétique à longueur d'onde millimétrique sur la croissance bactérienne

The study on the growth of E. coli cells under low power microwave irradiation the frequency of which can vary between 70 and 75 GHz, demonstrates that in the neighbourhood of 70.5 and 73 GHz a slowdown of growth is most frequently observed. On the contrary, the survival is not altered as long as the power of the irradiation is low. The irradiation does not induce lesions in DNA, whatever the frequency may be.

Thermal action of 2.45 GHz microwaves on the cytoplasm of Chinese hamster cells.

In order to demonstrate possible specific effects of microwaves at the cellular level V-79 Chinese hamster cells were exposed to 2.45-GHz radiation at power levels of 20-200 mW/cm2 and at specific absorption rates of 10-100 mW/g. Intracellular cytoplasmic changes were observed by fluorescence polarization using a method based on the intracellular enzymatic hydrolysis of nonfluorescent fluorescein diacetate (FDA). At levels of absorbed energy below 90 J/g, modifications of microviscosity and mitochondrial state were absent, but a slight stimulation of enzymatic hydrolysis of FDA was observed which may be explained by microwave-induced alterations of cellular membranes possibly due to differences in heating pattern of microwaves compared to water-bath heating. At levels of absorbed energy above 90 J/g, the decrease of enzymatic hydrolysis of FDA, increase in degree of polarization, and increase of permeation of the fluorescent marker correlated well with the decrease in cell viability as measured by the exclusion of trypan blue. At equal absorbed energy, microwaves were found to exert effects comparable to classical heating except that permeation was slightly more affected by microwave than by classical heating. This suggests that membrane alteration produced by microwaves might differ from those induced by classical heating or that microwaves may have heated the membrane to higher temperatures than did classical heating.