Michèle Dardalhon

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.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Organisms growing in aerobic environments must cope with Reactive Oxygen Species (ROS). Although ROS damage all the cellular macromolecules, they play a central role in a range of biological processes requiring a tight control of redox homeostasis. It is achieved by antioxidant systems involving a large collection of enzymes that scavenge or degrade the ROS produced endogenously during cell growth. In addition to this enzymatic protection against ROS, cells also contain small antioxidant molecules, such as glutathione (GSH). With an intracellular concentration between 1 and 10mM, GSH is the most abundant non-protein thiol in the cell and is considered as the major redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing different amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear DNA and nuclear functions are protected against oxidative injury, as exemplified by low mutation frequency, moderate histone carbonylation, activation of the checkpoint kinase Rad53 and of the H2O2 transcriptional response. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Redox-sensitive YFP sensors for monitoring dynamic compartment-specific glutathione redox state

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes.

Peroxiredoxin 1 knockdown potentiates β-lapachone cytotoxicity through modulation of reactive oxygen species and mitogen-activated protein kinase signals

Peroxiredoxin (Prx) 1 is a member of the thiol-specific peroxidases family and plays diverse roles such as H2O2 scavenger, redox signal transducer and molecular chaperone. Prx1 has been reported to be involved in protecting cancer cells against various therapeutic challenges. We investigated how modulations of intracellular redox system affect cancer cell sensitivity to reactive oxygen species (ROS)-generating drugs. We observed that stable and transient Prx1 knockdown significantly enhanced HeLa cell sensitivity to β-lapachone (β-lap), a potential anticancer agent. Prx1 knockdown markedly potentiated 2 µM β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 dependent and associated with a decrease in poly(ADP-ribose) polymerase 1 protein levels, phosphorylation of JNK, p38 and Erk proteins in mitogen-activated protein kinase (MAPK) pathways and a decrease in thioredoxin 1 (Trx1) protein levels. Trx1 serves as an electron donor for Prx1 and is overexpressed in Prx1 knockdown cells. Based on the fact that Prx1 is a major ROS scavenger and a partner of at least ASK1 and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through combined action of accumulation of ROS and enhancement of MAPK pathway activation, leading to cell apoptosis. These data support the view that modulation of intracellular redox state could be an alternative approach to enhance cancer cell sensitivity to ROS-generating drugs or to overcome some types of drug resistance.

Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae.

Abstract Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 – 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5′ region of the ARG4 gene twice as often as the Bgl site at the 3′ end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5′ region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5′ region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.

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

DETECTION OF PYRIMIDINE DIMERS AND MONOADDUCTSINDUCED BY 7‐METHYLPYRIDO(3,4‐c) PSORALEN AND UVA IN CHINESE HAMSTER V79 CELLS BY ENZYMATIC CLEAVAGE AND HIGH‐PRESSURE LIQUID CHROMATOGRAPHY

Abstract 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.

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

SummaryThe 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 crosslinks. 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.

Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a CanR mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the CanR mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.