Effects of ultraviolet radiation on the replication of DNA in higher eukaryotes

Le rayonnement ultraviolet (UV) émis par le soleil et qui atteint la peau de chaque individu est composé majoritairement de photons UVA (λ de 315 à 400 nm), le reste (5 à 10 %) étant composé d’UVB les plus longs (λ de 300 à 315 nm), car les radiations de longueur d’onde 300nm, c’est-à-dire les plus toxiques en terme de santé humaine, sont absorbées par la couche d’ozone stratosphérique. Contrairement aux UVB, les radiations UVA sont faiblement absorbées par l’ADN et de fait, génèrent peu de dimères cyclobutaniques de pyrimidines. Néanmoins, un des problèmes majeurs posés par une exposition aux UVA tient à ce que ce rayonnement excite certains composés endogènes photosensibles, inducteurs de la production d’espèces réactives de l’oxygène (ROS) qui peuvent alors endommager les composants cellulaires tels que les lipides,les acides nucléiques et les protéines. De ce fait, si les UVB restent le facteur étiologique majeur contribuant à la cancérogenèse cutanée photoinduite, un rôle des UVA, via la production de ROS, semble également émerger. Des précédents travaux obtenus au laboratoire ont montré que le rayonnement UVA ralentit la réplication de l’ADN, indépendamment de l’activation des points de contrôle du cycle cellulaire. Les auteurs ont émis l’hypothèse que les UVA, via l’oxydation des protéines, pouvaient altérer la machinerie de réplication. Mon travail de thèse a donc consisté à tenter de préciser le mécanisme qui gouverne ce retard de la réplication de l’ADN induit par les UVA dans les cellules de mammifères.Pour étudier au niveau moléculaire les effets des UVA sur la réplication, nous avons tout d’abord mis en place et utilisé au laboratoire la technique du peignage moléculaire (DNA combing) qui permet de mesurer divers paramètres de la réplication. Ainsi, nous montrons que le rayonnement UVA inhibe immédiatement et transitoirement les vitesses de fourches alors que l’inhibition sur l’initiation des origines est plus prolongée. Dans le cadre d’une collaboration, nous montrons également que les radiations UVA induisent une diminution modeste et transitoire du pool de dNTPs intracellulaires. La complémentation en ribonucléosides ne semble pas suffisante pour restaurer une vélocité normale de fourches immédiatement après UVA, ni la réplication dans sa totalité. En parallèle, nous observons l’oxydation réversible de la sous-unité R1 de la ribonucléotide réductase impliquée dans la biosynthèse des dNTPs. Bien que cette oxydation ne puisse expliquer la baisse transitoire du pool de nucléotides après UVA, nous ne pouvons pas exclure que d’autres formes d’oxydation de la RNR puissent affecter son activité.La présence d’azide de sodium (NaN3) au cours de l’irradiation UVA prévient le retard réplicatif, limite l’oxydation de la sous-unité R1 et la diminution du pool de dNTPs, ce qui démontre que ce retard de réplication est totalement dépendant des ROS, principalement de l’oxygène singulet généré pendant l’irradiation.L’ensemble de nos résultats indiquent que les UVA affectent le processus de réplication en modifiant non seulement la vélocité des fourches mais également l’initiation des origines de réplication. Puisqu’une perturbation de la réplication est une cause majeure d’instabilité génétique, il reste à déterminer si, dans nos conditions expérimentales, les radiations UVAfavorisent cette instabilité. Enfin, nous pensons que la ou les cibles des ROS induites par les UVA sont essentiellement cytosoliques et que le mécanisme conduisant à l’inhibition de la réplication n’est pas spécifique de ces ROS mais pourrait s’observer en utilisant d’autres types de stress oxydant.The solar UV radiation that reaches the earth’s surface is composed of 10 % UVB (280–320 nm) and 90 % UVA (320–400 nm) the main toxic radiations (wavelengths below 300 nm) being blocked by the stratospheric ozone. Unlike UVB, the UVA component of solar radiation is weakly absorbed by DNA. Nevertheless, one of major problems due to UVA exposure is the production of reactive oxygen species (ROS) through the interaction with endogenous and exogenous chromophores. These ROS cause damage to DNA, lipids and proteins. Even if UVB remains the major etiological factor known to be implicated in photoinduced cutaneous carcinogenesis, a novel role for UVA via the production of ROS seems to emerge. In our lab, previous works have provided evidence that exposure of mammalian cells to UVA-induced ROS led to delayed S-phase and reduced DNA synthesis, by a yet unknown process, which does not require a functional DNA damage checkpoint response, despite ATM-, ATR-, p38-dependent pathways activation. The authors proposed that inhibition of DNA replication is due to impaired replication fork progression and/or origins activation, as a consequence of UVA-induced oxidative damage to proteins rather than to DNA. The project for my PhD thesis is to better understand the mechanism underlying this UVA-induced slowdown of DNA replication in human cells.To study at the molecular level the effects of UVA on DNA replication, we used the DNA combing methodology. This technique allows measurement of the fork velocity and of the origins density. We show that UVA-induced ROS inhibit immediately after irradiation, but transiently, the progression of replication forks, while the inhibition on the initiation of originslasts longer. By HPLC-MS, we show that UVA radiation induces a moderate and transient decrease of the level of each intracellular dNTP. The supply of ribonucleosides doesn’t seem to be sufficient to restore neither a normal forks velocity immediately post-UVA nor the overall slowdown of DNA replication. In addition, we observe a reversible oxidation of the subunit R1 of ribonucleotide reductase, an enzyme which is involved in dNTPs biosynthesis. This oxidation cannot explain the transient reduction of dNTPs pool after UVA exposure, but other types of RNR oxidative modification could affect its activity. During UVA irradiation, the presence of the antioxidant sodium azide (NaN3) prevents the delay of DNA replication, limits the oxidation of the subunit R1 and the decrease of dNTPs pool. These results strongly suggest that the slowdown of DNA replication totally depends on ROS, in particular on singlet oxygen production induced by UVA.Altogether, our data indicate that UVA irradiation affects the process of DNA replication by modifying the forks velocity and the activation of origins. As DNA replication impairment is a major cause of genetic instability, it is of importance to determine if UVA irradiation leads to this instability in our experimental conditions. Finally, we suspect that the target of UVAinduced ROS is essentially cytosolic and that the mechanism driving the inhibition of replication is not specific of UVA-induced ROS, but could be also observed with other types of oxidative stress

Effets du rayonnement ultraviolet a sur la réplication de l’adn chez les eucaryotes supérieurs

The solar UV radiation that reaches the earth’s surface is composed of 10 % UVB (280–320 nm) and 90 % UVA (320–400 nm) the main toxic radiations (wavelengths below 300 nm) being blocked by the stratospheric ozone. Unlike UVB, the UVA component of solar radiation is weakly absorbed by DNA. Nevertheless, one of major problems due to UVA exposure is the production of reactive oxygen species (ROS) through the interaction with endogenous and exogenous chromophores. These ROS cause damage to DNA, lipids and proteins. Even if UVB remains the major etiological factor known to be implicated in photoinduced cutaneous carcinogenesis, a novel role for UVA via the production of ROS seems to emerge. In our lab, previous works have provided evidence that exposure of mammalian cells to UVA-induced ROS led to delayed S-phase and reduced DNA synthesis, by a yet unknown process, which does not require a functional DNA damage checkpoint response, despite ATM-, ATR-, p38-dependent pathways activation. The authors proposed that inhibition of DNA replication is due to impaired replication fork progression and/or origins activation, as a consequence of UVA-induced oxidative damage to proteins rather than to DNA. The project for my PhD thesis is to better understand the mechanism underlying this UVA-induced slowdown of DNA replication in human cells.To study at the molecular level the effects of UVA on DNA replication, we used the DNA combing methodology. This technique allows measurement of the fork velocity and of the origins density. We show that UVA-induced ROS inhibit immediately after irradiation, but transiently, the progression of replication forks, while the inhibition on the initiation of originslasts longer. By HPLC-MS, we show that UVA radiation induces a moderate and transient decrease of the level of each intracellular dNTP. The supply of ribonucleosides doesn’t seem to be sufficient to restore neither a normal forks velocity immediately post-UVA nor the overall slowdown of DNA replication. In addition, we observe a reversible oxidation of the subunit R1 of ribonucleotide reductase, an enzyme which is involved in dNTPs biosynthesis. This oxidation cannot explain the transient reduction of dNTPs pool after UVA exposure, but other types of RNR oxidative modification could affect its activity. During UVA irradiation, the presence of the antioxidant sodium azide (NaN3) prevents the delay of DNA replication, limits the oxidation of the subunit R1 and the decrease of dNTPs pool. These results strongly suggest that the slowdown of DNA replication totally depends on ROS, in particular on singlet oxygen production induced by UVA.Altogether, our data indicate that UVA irradiation affects the process of DNA replication by modifying the forks velocity and the activation of origins. As DNA replication impairment is a major cause of genetic instability, it is of importance to determine if UVA irradiation leads to this instability in our experimental conditions. Finally, we suspect that the target of UVAinduced ROS is essentially cytosolic and that the mechanism driving the inhibition of replication is not specific of UVA-induced ROS, but could be also observed with other types of oxidative stress.Le rayonnement ultraviolet (UV) émis par le soleil et qui atteint la peau de chaque individu est composé majoritairement de photons UVA (λ de 315 à 400 nm), le reste (5 à 10 %) étant composé d’UVB les plus longs (λ de 300 à 315 nm), car les radiations de longueur d’onde 300nm, c’est-à-dire les plus toxiques en terme de santé humaine, sont absorbées par la couche d’ozone stratosphérique. Contrairement aux UVB, les radiations UVA sont faiblement absorbées par l’ADN et de fait, génèrent peu de dimères cyclobutaniques de pyrimidines. Néanmoins, un des problèmes majeurs posés par une exposition aux UVA tient à ce que ce rayonnement excite certains composés endogènes photosensibles, inducteurs de la production d’espèces réactives de l’oxygène (ROS) qui peuvent alors endommager les composants cellulaires tels que les lipides,les acides nucléiques et les protéines. De ce fait, si les UVB restent le facteur étiologique majeur contribuant à la cancérogenèse cutanée photoinduite, un rôle des UVA, via la production de ROS, semble également émerger. Des précédents travaux obtenus au laboratoire ont montré que le rayonnement UVA ralentit la réplication de l’ADN, indépendamment de l’activation des points de contrôle du cycle cellulaire. Les auteurs ont émis l’hypothèse que les UVA, via l’oxydation des protéines, pouvaient altérer la machinerie de réplication. Mon travail de thèse a donc consisté à tenter de préciser le mécanisme qui gouverne ce retard de la réplication de l’ADN induit par les UVA dans les cellules de mammifères.Pour étudier au niveau moléculaire les effets des UVA sur la réplication, nous avons tout d’abord mis en place et utilisé au laboratoire la technique du peignage moléculaire (DNA combing) qui permet de mesurer divers paramètres de la réplication. Ainsi, nous montrons que le rayonnement UVA inhibe immédiatement et transitoirement les vitesses de fourches alors que l’inhibition sur l’initiation des origines est plus prolongée. Dans le cadre d’une collaboration, nous montrons également que les radiations UVA induisent une diminution modeste et transitoire du pool de dNTPs intracellulaires. La complémentation en ribonucléosides ne semble pas suffisante pour restaurer une vélocité normale de fourches immédiatement après UVA, ni la réplication dans sa totalité. En parallèle, nous observons l’oxydation réversible de la sous-unité R1 de la ribonucléotide réductase impliquée dans la biosynthèse des dNTPs. Bien que cette oxydation ne puisse expliquer la baisse transitoire du pool de nucléotides après UVA, nous ne pouvons pas exclure que d’autres formes d’oxydation de la RNR puissent affecter son activité.La présence d’azide de sodium (NaN3) au cours de l’irradiation UVA prévient le retard réplicatif, limite l’oxydation de la sous-unité R1 et la diminution du pool de dNTPs, ce qui démontre que ce retard de réplication est totalement dépendant des ROS, principalement de l’oxygène singulet généré pendant l’irradiation.L’ensemble de nos résultats indiquent que les UVA affectent le processus de réplication en modifiant non seulement la vélocité des fourches mais également l’initiation des origines de réplication. Puisqu’une perturbation de la réplication est une cause majeure d’instabilité génétique, il reste à déterminer si, dans nos conditions expérimentales, les radiations UVAfavorisent cette instabilité. Enfin, nous pensons que la ou les cibles des ROS induites par les UVA sont essentiellement cytosoliques et que le mécanisme conduisant à l’inhibition de la réplication n’est pas spécifique de ces ROS mais pourrait s’observer en utilisant d’autres types de stress oxydant

Effets du rayonnement ultraviolet a sur la réplication de l'adn chez les eucaryotes supérieurs

Le rayonnement ultraviolet (UV) émis par le soleil et qui atteint la peau de chaque individu est composé majoritairement de photons UVA ( de 315 à 400 nm), le reste (5 à 10 %) étant composé d UVB les plus longs ( de 300 à 315 nm), car les radiations de longueur d onde 300nm, c est-à-dire les plus toxiques en terme de santé humaine, sont absorbées par la couche d ozone stratosphérique. Contrairement aux UVB, les radiations UVA sont faiblement absorbées par l ADN et de fait, génèrent peu de dimères cyclobutaniques de pyrimidines. Néanmoins, un des problèmes majeurs posés par une exposition aux UVA tient à ce que ce rayonnement excite certains composés endogènes photosensibles, inducteurs de la production d espèces réactives de l oxygène (ROS) qui peuvent alors endommager les composants cellulaires tels que les lipides,les acides nucléiques et les protéines. De ce fait, si les UVB restent le facteur étiologique majeur contribuant à la cancérogenèse cutanée photoinduite, un rôle des UVA, via la production de ROS, semble également émerger. Des précédents travaux obtenus au laboratoire ont montré que le rayonnement UVA ralentit la réplication de l ADN, indépendamment de l activation des points de contrôle du cycle cellulaire. Les auteurs ont émis l hypothèse que les UVA, via l oxydation des protéines, pouvaient altérer la machinerie de réplication. Mon travail de thèse a donc consisté à tenter de préciser le mécanisme qui gouverne ce retard de la réplication de l ADN induit par les UVA dans les cellules de mammifères.Pour étudier au niveau moléculaire les effets des UVA sur la réplication, nous avons tout d abord mis en place et utilisé au laboratoire la technique du peignage moléculaire (DNA combing) qui permet de mesurer divers paramètres de la réplication. Ainsi, nous montrons que le rayonnement UVA inhibe immédiatement et transitoirement les vitesses de fourches alors que l inhibition sur l initiation des origines est plus prolongée. Dans le cadre d une collaboration, nous montrons également que les radiations UVA induisent une diminution modeste et transitoire du pool de dNTPs intracellulaires. La complémentation en ribonucléosides ne semble pas suffisante pour restaurer une vélocité normale de fourches immédiatement après UVA, ni la réplication dans sa totalité. En parallèle, nous observons l oxydation réversible de la sous-unité R1 de la ribonucléotide réductase impliquée dans la biosynthèse des dNTPs. Bien que cette oxydation ne puisse expliquer la baisse transitoire du pool de nucléotides après UVA, nous ne pouvons pas exclure que d autres formes d oxydation de la RNR puissent affecter son activité.La présence d azide de sodium (NaN3) au cours de l irradiation UVA prévient le retard réplicatif, limite l oxydation de la sous-unité R1 et la diminution du pool de dNTPs, ce qui démontre que ce retard de réplication est totalement dépendant des ROS, principalement de l oxygène singulet généré pendant l irradiation.L ensemble de nos résultats indiquent que les UVA affectent le processus de réplication en modifiant non seulement la vélocité des fourches mais également l initiation des origines de réplication. Puisqu une perturbation de la réplication est une cause majeure d instabilité génétique, il reste à déterminer si, dans nos conditions expérimentales, les radiations UVAfavorisent cette instabilité. Enfin, nous pensons que la ou les cibles des ROS induites par les UVA sont essentiellement cytosoliques et que le mécanisme conduisant à l inhibition de la réplication n est pas spécifique de ces ROS mais pourrait s observer en utilisant d autres types de stress oxydant.The solar UV radiation that reaches the earth s surface is composed of 10 % UVB (280 320 nm) and 90 % UVA (320 400 nm) the main toxic radiations (wavelengths below 300 nm) being blocked by the stratospheric ozone. Unlike UVB, the UVA component of solar radiation is weakly absorbed by DNA. Nevertheless, one of major problems due to UVA exposure is the production of reactive oxygen species (ROS) through the interaction with endogenous and exogenous chromophores. These ROS cause damage to DNA, lipids and proteins. Even if UVB remains the major etiological factor known to be implicated in photoinduced cutaneous carcinogenesis, a novel role for UVA via the production of ROS seems to emerge. In our lab, previous works have provided evidence that exposure of mammalian cells to UVA-induced ROS led to delayed S-phase and reduced DNA synthesis, by a yet unknown process, which does not require a functional DNA damage checkpoint response, despite ATM-, ATR-, p38-dependent pathways activation. The authors proposed that inhibition of DNA replication is due to impaired replication fork progression and/or origins activation, as a consequence of UVA-induced oxidative damage to proteins rather than to DNA. The project for my PhD thesis is to better understand the mechanism underlying this UVA-induced slowdown of DNA replication in human cells.To study at the molecular level the effects of UVA on DNA replication, we used the DNA combing methodology. This technique allows measurement of the fork velocity and of the origins density. We show that UVA-induced ROS inhibit immediately after irradiation, but transiently, the progression of replication forks, while the inhibition on the initiation of originslasts longer. By HPLC-MS, we show that UVA radiation induces a moderate and transient decrease of the level of each intracellular dNTP. The supply of ribonucleosides doesn t seem to be sufficient to restore neither a normal forks velocity immediately post-UVA nor the overall slowdown of DNA replication. In addition, we observe a reversible oxidation of the subunit R1 of ribonucleotide reductase, an enzyme which is involved in dNTPs biosynthesis. This oxidation cannot explain the transient reduction of dNTPs pool after UVA exposure, but other types of RNR oxidative modification could affect its activity. During UVA irradiation, the presence of the antioxidant sodium azide (NaN3) prevents the delay of DNA replication, limits the oxidation of the subunit R1 and the decrease of dNTPs pool. These results strongly suggest that the slowdown of DNA replication totally depends on ROS, in particular on singlet oxygen production induced by UVA.Altogether, our data indicate that UVA irradiation affects the process of DNA replication by modifying the forks velocity and the activation of origins. As DNA replication impairment is a major cause of genetic instability, it is of importance to determine if UVA irradiation leads to this instability in our experimental conditions. Finally, we suspect that the target of UVAinduced ROS is essentially cytosolic and that the mechanism driving the inhibition of replication is not specific of UVA-induced ROS, but could be also observed with other types of oxidative stress.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

UVA radiation (320-400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

<div><p>In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our <i>in silico</i> prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.</p></div

Depletion of intracellular GSH/GSSG pool prevents hXRCC3 oxidation induced by UVA_MEMi.

<p>CXR3 cells were pre-incubated for 24 h in culture medium containing or not 0.5 mM BSO. Thereafter, BSO-treated cells were complemented or not with 2 mM GSH-dEE or GSH-mEE for 1 h. (<b>A</b>) Measurement of GSH level in cells. Values are expressed as % of GSH relative to control cells (–BSO) and results are the mean ± SD of 3 independent experiments. Cells treated as described in panel A were exposed to 160 kJ/m² UVA_MEMi and lysed immediately post radiation in buffer containing either 10 mM NEM (<b>B</b>) or 4 mM malPEG (<b>D</b>). (<b>C</b>) CXR3, irs1SF and XR3/S cells were exposed to 160 kJ/m² UVA_MEMi and immediately lysed in buffer containing 4 mM malPEG. The star (*) indicates non specific cross-reactivity of anti-XRCC3 antibody. In (C) and (D), the samples were analysed in reducing condition (+ßME). Myc-tagged XR3/S protein was detected using anti-myc antibody, and S-glutathionylated proteins using anti-GSH antibody. Actin was used as loading control. ßME: ß-mercaptoethanol. (<b>E</b>) GSH-proficient (−BSO and +BSO +GSH-dEE) and deficient (+BSO) cells were irradiated at 160 kJ/m² UVA_MEMi and cell viability was assessed 24 h post treatment by MTT assay. Values are the mean +/− SD of 5 independent experiments. Statistical analysis was performed using ANOVA with TUKEY’s post test. * P<0.05; ** P<0.01.</p

Spatial cysteine distribution in human XRCC3.

<p>(A) Sequence alignment of the archaeal RadA from <i>Sulfolobus solfataricus</i>, the archaeal RadA from <i>Methanococcus voltae</i>, the RAD51 protein of <i>Saccharomyces cerevisiae</i> and the human XRCC3 (Hum. Xrcc3) performed with the program ClustalW. These enzymes share characteristic features like the L1 and L2 DNA putative binding regions, an ATP cap, a P-loop as well as conserved residues involved in ATP binding or ATPase activity. Conserved amino acid residues are shadowed in red. The positions of cysteine residues in the four structures are shadowed in yellow. The missing residues in the template crystal structures are underlined and in italic. (<b>B</b>) Stereo ribbon diagram showing the positions of the 8 cysteine residues on the modeled structure of the human XRCC3. The Cα positions of Cys86, Cys141, Cys193, Cys221, and Cys328, potentially solvent accessible, are figured in yellow. In red are the positions of Cys121 and Cys255, both buried in the structure of XRCC3. The Cα position of residue Cys310 is in green. Panels B was made with Pymol molecular visualisation system (DeLano Scientific LLC).</p

N-acetyl-L-cystein prevents hXRCC3 oxidation and its relocalization at the chromatin in response to MN.

<p>(<b>A</b>) CXR3 cells were exposed to 25 and 50 µM MN for 10 min at 37°C. S-glutathionylated proteins and oxidation of hXRCC3 protein were analyzed by Western blot at different time points after treatment. XRCC3, prot-SSG, and actin were detected using anti-XRCC3, anti-GSH, or anti-actin antibodies, respectively. (<b>B</b>) CXR3 cells were exposed to 50 µM MN in the presence or not of 10 mM NAC and hXRCC3 protein oxidation was analyzed at different time points after treatment by Western blot. (<b>C</b>) CXR3 cells were exposed to 50 and 100 µM MN for 1h in the presence or not of 10 mM NAC. (<b>D</b>) Cells expressing wild-type (CXR3, XR3/C) or mutated (XR3/S, XR3/S86 and XR3/S328) hXRCC3 were exposed to 100 µM MN for 1h. In (<b>C</b>) and (<b>D</b>), the soluble (Sol. frac.) and chromatine (Chr. frac.) fractions were recovered immediately post treatment. hXRCC3 and hXRCC3-myc tagged proteins were analysed by Western blot using anti-XRCC3 antibody. GAPDH and Lamin A/C were used as loading control. ßME: ß-mercaptoethanol. (<b>E</b>) XRCC3 proficient (CXR3) and deficient (irs1SF) cells were exposed to 100 µM MN for 10 and 60 min. Cell viability was assessed 24 h post treatment by MTT assay. Values are the mean +/− SD of 5 independent experiments.</p

GSH/GSSG pool is not required for hXRCC3 oxidation in CHO cells in response to oxidants other than UVA_MEMi.

<p>CXR3 cells treated or not with BSO were exposed to increasing concentrations of (<b>A</b>) tButH₂O₂ for 30 min, (<b>B</b>) menadione (MN) for 10 min, (<b>C</b>) UVA+Rufloxacine (UVA_RFX), (<b>D</b>) 1,4-dinitrochlorobenzene (DNCB) for 30 min and (<b>E</b>) diamide for 30 min. Immediately post treatments, cells were lysed in buffer containing 10 mM NEM and protein extracts analysed by Western blot in non-reducing (-ßME) and reducing (+ßME) conditions. Prot-SSG adducts, hXRCC3, PCNA and actin were detected using anti-GSH, anti-XRCC3, anti-PCNA, and anti-actin antibodies, respectively. PCNA^mono = monomeric PCNA; PCNA^tri = covalently bound trimeric PCNA. ßME: ß-mercaptoethanol. (<b>F</b>) CXR3, XR3/C and XR3/S cells were exposed to 1 mM diamide for 30 min, and soluble (Sol. frac.) and chromatine (Chr. frac.) fractions were recovered. hXRCC3 and hXRCC3-myc tagged proteins were detected using anti-XRCC3 antibody. GAPDH and Lamin A/C were used as loading control.</p

Reversible oxidation of hXRCC3.

<p>CXR3 cells were exposed to different doses of UVA_MEMi and total soluble protein extracts were prepared either immediately (<b>A</b>) or at various times post radiation (<b>B</b>). irs1SF cells were used as negative control for XRCC3 expression. Cells were lysed on ice in lysis buffer containing 10 mM N-ethylmaleimide (NEM). Protein extracts in non reductive (-ßME) and reductive (+ßME) conditions were loaded on 9% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were probed with rabbit polyclonal anti-XRCC3 antibody. (*) indicates non-specific cross-reactivity of the antibody. Actin was used as loading control. ßME: ß-mercaptoethanol.</p