Radiat Environ Biophys DOI 10.1007/s00411-014-0569-y

ORIGINAL PAPER

UV radiation effects on a DNA repair enzyme: conversion of a [4Fe–4S]2+ cluster into a [2Fe–2S]2+ Filipe Folgosa • Ineˆs Camacho • Daniela Penas • Ma´rcia Guilherme • Joa˜o Fro´is • Paulo A. Ribeiro Pedro Tavares • Alice S. Pereira

•

Received: 21 March 2014 / Accepted: 17 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Organisms are often exposed to different types of ionizing radiation that, directly or not, will promote damage to DNA molecules and/or other cellular structures. Because of that, organisms developed a wide range of response mechanisms to deal with these threats. Endonuclease III is one of the enzymes responsible to detect and repair oxidized pyrimidine base lesions. However, the effect of radiation on the structure/function of these enzymes is not clear yet. Here, we demonstrate the effect of UV-C radiation on E. coli endonuclease III through several techniques, namely UV–visible, fluorescence and Mo¨ssbauer spectroscopies, as well as SDS-PAGE and electrophoretic mobility shift assay. We demonstrate that irradiation with a UV-C source has dramatic consequences on the absorption, fluorescence, structure and functionality of the protein, affecting its [4Fe–4S] cluster and its DNAbinding ability, which results in its inactivation. An UV-C radiation-induced conversion of the [4Fe–4S]2? into a [2Fe–2S]2? was observed for the first time and proven by Mo¨ssbauer and UV–visible analysis. This work also shows that the DNA-binding capability of endonuclease III is

Electronic supplementary material The online version of this article (doi:10.1007/s00411-014-0569-y) contains supplementary material, which is available to authorized users. F. Folgosa  I. Camacho  D. Penas  M. Guilherme  J. Fro´is  P. Tavares  A. S. Pereira (&) REQUIMTE/CQFB, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal e-mail: [email protected] F. Folgosa  P. A. Ribeiro CEFITEC, Departamento de Fı´sica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

highly dependent of the nuclearity of the endogenous iron– sulfur cluster. Thus, from our point of view, in a cellular context, these results strengthen the argument that cellular sensitivity to radiation can also be due to loss of radiationinduced damage repair ability. Keywords BER enzymes  DNA repair  Endonuclease III  Ionizing radiation  Oxidative stress  Iron–sulfur proteins

Introduction The genome is constantly exposed to agents, exogenous and endogenous, that can promote damage to DNA. These lesions arise from three main causes: (1) environmental agents, such as UV components of sunlight, ionizing radiation and a variety of genotoxic chemicals (such as cigarette smoke, alcohol, or biocides and pesticides); (2) (by)products of normal cellular metabolism, such as reactive oxygen species (ROS); and (3) spontaneous degradation under physiological conditions (oxidation, ethylation and hydrolysis of bases). The outcome of DNA damage is diverse and generally adverse; long-term effects result on irreversible mutations that contribute to oncogenesis. As a consequence, cells have developed sophisticated DNA repair systems that cover most of the lesions inflicted. These systems are highly conserved from prokaryotes to eukaryotes. Base excision repair (BER) is one of the major systems that repair the DNA damage caused by ionizing radiation, removing damaged bases. It acts on subtle, non-bulky, DNA lesions through a multistep mechanism that involves the participation of several enzymes (Scharer 2003; Hoeijmakers 2001). The first step in this pathway is catalyzed by DNA glycosylases that locate damaged or mismatched

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bases and excise them from the DNA helix, leaving an apurinic–apyrimidic site. Subsequent action of apurinic– apyrimidic endonucleases, DNA polymerases and ligases restores the correct nucleotide. Endonuclease III (EndoIII) is a ubiquitous BER enzyme that repairs oxidized pyrimidine base lesions. It is a bifunctional DNA N-glycosylase (base excision)/b-lyase (strand nicking) enzyme. The Escherichia (E.) coli EndoIII was the first DNA glycosylase discovered that contain a [4Fe–4S] cluster. Other DNA glycosylases with the same metal cofactor have been identified, including the E. coli mismatch-specific adenine glycosylase, MutY, a monofunctional enzyme that is involved in the repair of 7,8-dihydro-8-oxo-20 -desoxyguanine:adenine and G:A mispairs. Combination of kinetic, mutagenic and structural studies on E. coli MutY showed that although the cluster does not contribute significantly to the folding and stability of the protein, it is essential for DNA binding and catalysis (Porello et al. 1998). Electrochemistry data showed that binding to DNA shifts the redox potential of the [4Fe–4S]2?/3? couple and stabilizes the 3? oxidation state, leading to the proposal of a DNAmediated charge transport as a way to rapidly and efficiently search for DNA damaged sites in the genome (Boal et al. 2007; Lukianova and David 2005). Other studies carried out in DNA aerobic aqueous solutions indicated that UV-C radiation in aerobic aqueous solutions was able to promote damage to DNA molecules either directly or by inducing the formation of 1O2 and ROS (Santos et al. 2013; Wei et al. 1997). DNA damage caused by low-energy synchroton radiation, ranging from 7 to 150 eV, promoting single and double strand breaks in both dry and solution DNA plasmid was observed (Endres et al. 2004), pointing to free radical DNA chemistry damage (Long et al. 2003; Fink and Schonenberger 1999). In addition, the reduction in conductivity of DNA thin films, as a result of UV-C irradiation at 254 nm exposure, was associated with the concomitantly decrease in DNA number of phosphate groups (Gomes et al. 2009), which act as electron acceptors in the electron hopping between base pairs and DNA phosphate groups (Gomes et al. 2012). Wei and co-workers studies demonstrated the formation of DNA oxidative products such as 8-hydroxy-20 -deoxyguanosine (8-OHdG) when UV-C is used to irradiate purified calf thymus DNA samples (Zhang et al. 1997; Wei et al. 1997). Although ROS formation with UV-C irradiation in cellular conditions was not as significant as with UV-A or UV-B, according to Santos et al. (2013), these species are known to react with other biological molecules in the cell producing free radicals, which in turn can harm and modify many molecules, from DNA to proteins. Hydroxyl radicals, for example, may lead either to oxidative damage of DNA (through base oxidation, DNA lesions or DNA breaks) or of lipids and proteins. Proteins can be damaged by

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oxidative modifications of amino acid residues or peptide cleavage, resulting in malfunction of many enzymes (Mena et al. 2009; Lau et al. 2008; Sowa et al. 2006). Although the effects of UV-C and oxidative stress on DNA have been extensively studied (Silva et al. 2012; Zhang et al. 1997; Wei et al. 1997), the knowledge on the effect of this carcinogen on DNA repair enzymes, in particular on the Fe–S containing DNA glycosylases, such as EndoIII, is still limited. Whereas intact DNA repair is essential for a healthy organism, selective inhibition of DNA repair enzymes in malignant cells may greatly improve antitumor therapies. Therefore, the primary objective of the present work was to evaluate the effect induced by UV-C-mediated oxidative stress to E. coli endonuclease III and, in particular, to its [4Fe–4S] cluster. Spectroscopic and biochemical methods, such as UV–visible, fluorescence or Mo¨ssbauer spectroscopies, as well as electrophoretic methods (SDSPAGE and EMSA) were used to monitor not only the integrity of the [4Fe–4S] cluster but also the chemical modification of the primary structure on irradiated samples and their effect on the ability of the protein to bind DNA.

Materials and methods Cloning, expression and purification of recombinant E. coli Endonuclease III The overexpression vector was constructed using standard molecular biology methods. The gene encoding endonuclease III was amplified from E. coli DH5a genomic DNA by polymerase chain reaction (PCR) using two oligonucleotide primers homologous to the 50 and 30 ends, with a Nde I and a EcoR I restriction sites, respectively, for cloning into the pET-21c expression vector (Novagen). The construct was subsequently used to transform E. coli C43(DE3) competent cells (OverExpress). For protein expression, 5 mL of 2xYT medium containing 100 lg/mL ampicillin was inoculated with a single colony of E. coli C43(DE3) cells harboring the recombinant pET-21c–EndoIII construct. After growing for 8 h at 37 °C, at 220 rpm, 1 mL of this culture was used to inoculate 100 mL of 2xYT medium containing 100 lg/mL ampicillin. The culture was grown overnight at 37 °C at 220 rpm. This culture was subsequently used to inoculate 1 L of 2xYT medium containing 100 lg/mL ampicillin that was grown at 37 °C at 180 rpm, until an optical density at 600 nm of approximately 0.5 was reached. At this point, 0.1 mM Fe2SO4 was added, and protein expression was induced by addition of 0.2 mM of IPTG; cells were allowed to grow overnight at 20 °C. Cells were harvested by centrifugation, resuspended in 100 mM Tris–HCl buffer, pH 7.1 and lysed by sonication on a

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Labsonic M homogenizer, from Sartorius, that was equipped with a 20-mm diameter tip. The cells were intermittently sonicated on ice for 180 s with 90 s of cooling, in a total sonication time of 9 min at 100 W. The crude extract was cleared by low-speed centrifugation at 10,0009g for 20 min and at 138,0009g for 1 h and 30 min and at 4 °C to remove cell debris and membrane fraction, respectively. The supernatant was then dialyzed overnight at 4 °C against 100 mM Tris–HCl, pH 7.1. The soluble crude extract was then loaded onto a pre-packed Resource STM Fast Flow column (6 mL, GE Healthcare Life Sciences) previously equilibrated with 100 mM Tris–HCl, pH 7.1. Recombinant EndoIII (rEndoIII) was eluted with a linear gradient from 100 mM to 1 M Tris–HCl, pH 7.1. Fractions containing pure protein were pooled after SDS-PAGE analysis using a 4–12 % Bis–Tris precast gel (NuPAGEÒ Novex from Life Technologies) and by UV–visible spectroscopy (Evolution 300 UV–vis spectrophotometer from Thermo Scientific). The 57Fe-enriched protein was overexpressed following the same procedure as the 56Fe-protein but using minimum M9 as growth medium instead of 2xYT. When the optical density at 600 nm reached approximately 0.5, 0.1 mM 57Fe was added along with 0.2 mM of IPTG and cells were allowed to grow. Protein was purified according with the procedure described for the 56Fe-protein. Protein quantification and metal determination Pure rEndoIII samples were quantified using the Bicinchoninic Acid Kit (Sigma) and using bovine serum albumin as standard. Iron determination was performed according with previously published phenanthroline method (Tetlow and Wilson 1964) in which a pure protein fraction was reduced with L(?)-ascorbic acid sodium salt and incubated for 15 min with 8 M HCl and 80 % trichloroacetic acid. The sample was then centrifuged at 5,0009g and the pellet discarded. The supernatant was used to proceed with the iron quantification protocol in which triplicates of three sample dilutions were incubated with 0.3 % of 1,10–phenanthroline and 10 % of hydroxylamine hydrochloride for 10 min. Absorbance at 512 nm was measured and compared with the standard curve prepared in parallel with a commercial standard iron solution. UV irradiation studies To assess rEndoIII susceptibility to UV-C, irradiation assays were performed in a homemade irradiation box, equipped with a 5 W (1.1 W in UV-C, 254 nm) TUV PL-S 5 W/2P 1CT lamp (Philips). Protein samples were kept in UV-transparent cuvettes (UV-Cuvette micro, Brand) sealed with a rubber cap (Brand). Temperature inside the irradiation box was kept at 15 °C. Cuvettes containing protein

samples (400 lL of 6.18 lM of rEndoIII in 100 mM Tris– HCl, pH 7.1) were held over ice during all irradiation procedure. The irradiance (W/cm2) applied to each sample was measured with a HD2102.2 photo-radiometer (Delta Ohm) equipped with a LP471UVC probe (Delta Ohm). The irradiance was converted to radiation dose (J) by taking into account the exposed area (cm2) and the total exposure time (s), using the expression:

Dose ðJÞ ¼ irradiance W=cm2  exposed area cm2  time ðsÞ

ð1Þ

A table containing the radiation dose correspondent to each exposure time is supplied in Supplementary Table S1. 57 Fe-enriched protein samples were irradiated following the same protocol as the 56Fe-protein. UV–visible and SDS-PAGE studies Effects of UV-C radiation on rEndoIII-irradiated samples were assessed by UV–visible and SDS-PAGE. UV–visible spectra of rEndoIII samples were collected at the beginning and at the end of each irradiation assay. SDS-PAGE studies were performed using 4–12 % gradient NuPAGEÒ Bis– Tris pre-cast gel from (Life Technologies) that were run for 35 min at 200 V in 1x NuPAGEÒ SDS running buffer, stained with Coomassie Brilliant Blue R-250 and imaged with Safe ImagerTM in a Gel Logic 100 Imaging System. For densitometric quantification of the monomeric rEndoIII band as well as the dimer band, the electrophoretic image was processed using ImageJ v. 1.45S (http://rsbweb. nih.gov/ij/) (Schneider et al. 2012). Fluorescence studies of irradiated EndoIII Effect of UV-C radiation on rEndoIII structure was tested by steady-state fluorescence emission. This was used not only to detect tyrosine and tryptophan emission variation but also to evaluate dityrosine formation upon irradiation. Thus, after exposure to UV-C, protein samples were diluted to a final concentration of 2 lM, in 100 mM Tris–HCl pH 7.1, to avoid saturation of the emission spectra. Emission spectra were recorded in a Perkin-Elmer LS45 Luminescence Spectrometer between 310 and 500 nm for kExc = 275 nm and between 350 and 550 nm for kExc = 315 nm, with a slit width of 10 nm in both cases, in order to excite tyrosine/ tryptophan or dityrosine, respectively (Correia et al. 2012; Malencik and Anderson 2003; Lehrer and Fasman 1967). Mo¨ssbauer spectroscopy analysis Mo¨ssbauer spectra were recorded on a weak-field spectrometer operating at constant acceleration mode in

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transmission geometry. The zero velocity of the Mo¨ssbauer spectrum was referred to the centroid of the room-temperature spectrum of a metallic iron foil. The spectrometer was equipped with a Janis Closed Cycle He gas refrigerator cryostat with sample temperature range from 4.5 to 325 K. Mo¨ssbauer analysis was performed using the WMOSS software (SEE Co., Edina, MN, http://www.seeco.us/). Samples used for Mo¨ssbauer spectroscopy were dialyzed to 100 mM Tris–HCl pH 7.1 containing 100 mM NaCl and concentrated up to 0.6 mM in 57Fe. Electrophoretic mobility shift assays (EMSAs) The effect of UV-C irradiation on the DNA-binding ability of E. coli rEndoIII was tested by EMSA. Exposed and nonexposed (control) protein samples (from 0.04 lM up to 10 lM of rEndoIII) were incubated with non-exposed supercoiled plasmid DNA (10 nM pUC18, 2,686 bp) at different protein-pDNA ratios (from 4:1 up to 1,000:1) for 2 h, at room temperature, in 100 mM Tris–HCl, pH 7.1. Complex formation was then evaluated by 1 % agarose gel electrophoresis in 1 9 TAE buffer (40 mM Tris–acetate and 1 mM EDTA, pH 8.0), ran at 80 V at room temperature for 1 h 45 min. The results were visualized with SYBR Safe DNA gel stain and imaged with Safe ImagerTM in a Gel Logic 100 Imaging System. Free pDNA (10 nM) and rEndoIII (10 lM) samples were also assessed for control. To determine DNA-binding ability of rEndoIII, the electrophoretic images (triplicates) were processed (free pDNA and protein-DNA complex bands) using ImageJ v. 1.45S (http://rsbweb.nih.gov/ij/) (Schneider et al. 2012). For densitometric quantification, regions above the free supercoiled DNA were considered as protein-pDNA complex. The relative complex formation was plotted as a function of protein concentration and fitted to the Hill equation, f = fmax [EndoIII]n/(Kd ? [EndoIII]n), in which f is the fractional saturation, fmax corresponds to 100 % complex formation, [EndoIII] is the concentration of the binding protein, n is the Hill coefficient and Kd represents the macroscopic apparent dissociation constant and is a measure of the affinity of the protein to pDNA (Timoteo et al. 2012).

per protein was determined, which was in accordance with the expected 4 irons per protein from the [4Fe–4S] cluster. The UV–visible spectrum (Supplementary Fig. S1) of pure protein showed a protein peak at 280 nm and a broad absorption band with a maximum around 410 nm. The spectrum was similar to the one previously published by Asahara et al. (1989) and characteristic of oxidized [4Fe– 4S] cluster containing proteins (Staples et al. 1996; Flint et al. 1993). The apparent molecular mass of the recombinant protein monomer, determined by SDS-PAGE, was 23.4 ± 0.7 kDa, which was in agreement with the mass deduced from the amino acid sequence (23.6 kDa). UV–visible and SDS-PAGE studies DNA damage caused by UV-C radiation has been largely described in the literature (Silva et al. 2012; Zhang et al. 1997; Wei et al. 1997; Ramirez-Guadiana et al. 2012; Pfeifer et al. 2005). In this work, we aimed to evaluate the effect of this type of radiation on a DNA repair enzyme. Therefore, the UV-C radiation effect was evaluated by exposing rEndoIII samples for different periods (from 30 s to 120 min) to an UV-C light source (emission band at 254 nm). Figure 1 shows the UV–visible spectra of irradiated samples, revealing a decrease in the intensity of the *410 nm absorption band, characteristic of the [4Fe– 4S]2? cluster with increasing irradiation exposure. Simultaneously, the intensity of the 320-nm band (attributed to charge transfer transitions associated with a tetrahedrally coordinated iron centers with cysteinate ligation) increases, and from 30 to 45 min of exposure, a smooth shoulder was

Results Protein characterization Recombinant EndoIII was purified to homogeneity as described in the experimental section. Metal content and protein determination were assessed using BCA and Lowry assays and the 1,10-phenanthroline method (Lowry et al. 1951; Tetlow and Wilson 1964). A ratio of 3.9 ± 0.7 irons

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Fig. 1 UV–visible spectra of UV-C-irradiated rEndoIII. rEndoIII samples (5 lM), in 100 mM Tris–HCl, pH 7.1, were UV-C irradiated for different times, from 30 s (3 mJ) up to 120 min (788 mJ). Arrows point to changes in the absorbance. The inset represents the irradiation kinetic trace of [4Fe–4S]2? cluster absorbance band

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observed near 450 nm. This last observation was similar to the one described by Agarwalla and co-workers, when the [4Fe–4S]2? cluster of E. coli RumA is oxidized with ferricyanide, originating a [2Fe–2S]2? cluster (Agarwalla et al. 2004). After 80 min of exposure, total bleaching was observed. Thus, at this point, the disappearance of the visible component of the spectrum could be associated either with (1) the photoreduction of the [4Fe–4S] cluster, from the ?2 to the ?1 oxidation state, as previously described by Cunningham et al. (1989) or (2) denaturation of the protein and/ or disassembly of the [4Fe–4S] cluster (Girard et al. 2011; Wang and Wen 2010; Crack et al. 2008b; Agarwalla et al. 2004). The subunit composition of the same samples was assessed by SDS-PAGE. This experiment showed a decrease in the intensity of the EndoIII band with increasing irradiation time, from 10 to 120 min (Fig. 2). Also, a higher molecular mass band with apparent molecular mass of 44.1 ± 0.6 kDa was detected at irradiation times longer than 45 min. The appearance of this higher molecular mass band with approximately twice the apparent molecular mass of EndoIII may indicate the formation of covalent cross-links between two protein molecules. Dityrosine formation, for example, is one of the products of protein irradiation and it is well described in the literature (Correia et al. 2012; Borsarelli et al. 2012; Chakraborty et al. 2010). At longer irradiation times (6 h), both monomer and dimer bands disappear and a faint smear was observed. Fluorescence spectroscopic studies To investigate the hypothesis of a dityrosine cross-link formation, and thus covalent dimerization of the protein, irradiated samples were analyzed by steady-state fluorescence spectroscopy. In this case, we observed the buildup of the emission band with maximum around 420 nm when kExc = 315 nm was used, which was in accordance with what is described in the literature (Kungl et al. 1994; Chakraborty et al. 2010) to support the formation of dityrosine complexes (Fig. 3b). The formation of these complexes was detected after approximately 45 min of irradiation, while the maximum intensity was reached at 60 min of irradiation, stabilizing until 80 min and followed by a decrease in the emission band intensity. These observations were in accordance with the appearance of a protein band with twice the apparent molecular mass in the SDS-PAGE gel and concomitant loss of intensity of the monomer band. UV-C-induced denaturation of the protein would favor the exposure of tyrosine side chains residues promoting cross-linking, as well as the loss or structural modification of the iron–sulfur cluster, which in turn could

Fig. 2 Effects of radiation on rEndoIII assessed by SDS-PAGE. Lane 1 corresponds to the SeeBlue Pre-stained standard (Novex, Life Technologies); lanes 2–12 represent the protein irradiated for 0, 5 (3 mJ), 10 (66 mJ), 20 (131 mJ), 30 (197 mJ), 45 (296 mJ), 60 (394 mJ), 80 (526 mJ), 100 (657 mJ), 120 (788 mJ) and 360 (2,365 mJ) min, respectively. The gel (4–12 % gradient NuPAGEÒ Bis–Tris pre-cast gel from Life Technologies) was run for 35 min at 200 V and stained with Coomassie Brilliant Blue R-250. In the bottom image, the densitometric quantification of the monomer band (*23 kDa) and dimer band (*44 kDa) is presented. The molecular mass (in kDa) of each protein standard band is indicated on the left side of the figure

explain the modifications observed on the UV–visible spectrum of the protein upon UV irradiation. The formation of covalent bonds between tyrosine residues to form dityrosines derivatives upon exposure to radiation, or oxidative stress conditions, is well documented (Correia et al. 2012; Borsarelli et al. 2012; Chakraborty et al. 2010; Lim et al. 2007). The decrease of the fluorescence intensity in the samples exposed for longer UV-C irradiation times again indicates a higher degree of structural changes or protein aggregation. In fact, the 6-h-exposed sample showed a smear on the SDS-PAGE gel and the disappearance of the monomer band. In order to correlate this observation with the fluorescence emission of tyrosine and tryptophan residues and also to infer about overall polypeptide chain modifications, a second steady-state fluorescence spectroscopy study, using kExc = 275 nm, was performed (Fig. 3a). As expected, the emitted fluorescence is probably dominated by the contribution of the tryptophan residues as indicated by the observed emission maximum around 350 nm. The evolution of this emission band was not linear with the irradiation time, showing a bi-phase behavior: a strong increase in the first 2 min of irradiation followed by a decrease of the fluorescence emission intensity. Fluorescence emission of tryptophan residues in proteins depends on the polarity of the local environment; thus, the results obtained confirmed that several structural changes exist

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Fig. 3 Steady-state fluorescence study of UV-C-irradiated rEndoIII. a steady-state fluorescence emission spectra were collected using kEx = 275 nm. Inset represents the normalized fluorescence emission at 346 nm with irradiation time. For samples irradiated up to 2 min (13 mJ), an increase in emission intensity is observed. The fluorescence emission at this wavelength then decreases for irradiations longer than 2 min (13 mJ). The fluorescence emission trace was normalized by dividing each data point by the maximum emission value obtained at 2 min (13 mJ) exposure. b Steady-state fluorescence emission spectra were collected using kEx = 315 nm. Inset represents the normalized fluorescence emission at 420 nm with irradiation time. The buildup of an emission band with maximum around 420 nm is observable after 45 min (296 mJ) of irradiation indicating the formation of intermolecular dityrosine cross-links. The fluorescence emission trace was normalized by dividing each data point by the maximum emission value obtained at 80 min (526 mJ) exposure

Fig. 4 Low-field Mo¨ssbauer spectra of rEndoIII-irradiated samples. Spectra a–e are the Mo¨ssbauer spectra of the 0 (0 mJ), 20 (131 mJ), 45 (296 mJ), 80 (526 mJ) and 120 min (788 mJ) UV-C-irradiated protein samples, respectively. Spectra were acquired at 180 K with an applied field of 0.6 kG parallel to the c-beam. The line on the top of each spectrum represent the best fit and is the result of the superposition of different iron species with percentage contributions described on Table 1; the solid lines above each spectrum represent the [4Fe–4S]2? contribution, the dashed lines correspond to species I and the dash-dotted line represent species II. Spectrum at 180 K of the non-irradiated sample (0 min sample) was fitted with a single quadrupole doublet with DEQ = 1.06 ± 0.03 mm/s and d = 0.38 ± 0.02 mm/s. The following parameters: DEQI = 0.59 ± 0.03 mm/s and dI = 0.25 ± 0.02 mm/s and DEQII = 0.66 ± 0.01 mm/s and dII = 0.43 ± 0.03 mm/s were used to fit species I (assigned to [2Fe–2S]2? type cluster) and II (oxo/hidroxo-ferric polynuclear aggregates), respectively

Mo¨ssbauer spectroscopy studies upon irradiation. Moreover, the initial increase in emission intensity was associated to a structural change that occurs prior to [4Fe–4S]2? cluster conversion, while the later decrease in emission intensity follows a deeper structural change that was timely with the [2Fe–2S]2? appearance and subsequent oxo/hidroxo-ferric polynuclear aggregates formation (see UV–visible and Mo¨ssbauer spectroscopy section).

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As mentioned before, the [4Fe–4S] cluster is described in the literature to be necessary for DNA binding (Lukianova and David 2005). Therefore, Mo¨ssbauer spectroscopy was used to identify the iron species generated during UV-C irradiation of rEndoIII to later compare with the DNAbinding assays. This spectroscopy is particularly suited to these studies since all iron species can be accounted, i.e.,

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there is no silent iron forms, making possible to understand the fate of the [4Fe–4S] cluster upon radiation exposure. Four Mo¨ssbauer samples were prepared and irradiated from 20 min up to 120 min and a non-irradiated control. The low-field Mo¨ssbauer spectrum of the control sample at 4.2 K was best fitted with a single quadrupole doublet with DEQ = 1.18 ± 0.03 mm/s and d = 0.44 ± 0.01 mm/s, characteristic of [4Fe–4S]2? as previously described by Cunningham et al. (1989). Mo¨ssbauer spectra of all five samples recorded at 180 K are presented in Fig. 4. Besides the iron species observed in the control sample (spectrum A in Fig. 4), two other species, here after named species I and II, were detected. A 20-min exposure of the protein to radiation resulted in the formation of species I, that accounted to about 18 % of the total iron absorption. This species could be fitted with the following parameters: DEQI = 0.59 ± 0.03 mm/s and dI = 0.25 ± 0.02 mm/s. The second species, II, was detected after 80 min of irradiation and could be fitted with DEQII = 0.66 ± 0.01 mm/s and dII = 0.43 ± 0.03 mm/s. Despite that we cannot rule out that similar parameters can be attributed to a [3Fe–4S]?1 cluster (Huynh et al. 1980) or to the rubredoxin-type [FeCys4] (Yoo et al. 2000; Pereira et al. 2007), the parameters used to fit species I were also typical of a [2Fe–2S]2? cluster (Krebs et al. 2000), which was in accordance with the UV–visible data. Based on this observation, we favored the conversion of the [4Fe–4S]2? into a [2Fe–2S]2? state. A rubredoxin-type iron cluster in the oxidized state has very distinct features in the UV–visible spectrum that were not observed in our data (Moura et al. 1978). Moreover, the conversion, upon oxidation, of similar [4Fe–4S] clusters described in the literature usually yields small amounts of [3Fe–4S]1? clusters in favor of [2Fe–2S]2? clusters (Agarwalla et al. 2004; Cunningham et al. 1989). The parameters used to fit species II can be attributed to high-spin Fe3? (S = 5/2) most likely aggregated in the form of polynuclear particles, similar to amorphous mineral core of bacterial and plant ferritins or polymeric (Fe3?O6) systems as previously reported (Muh et al. 1997; Bottger et al. 2012). The Mo¨ssbauer absorption percentages of the three iron species, summarized in Table 1, changed with irradiation time. UV–visible of parallel samples was also performed at the same protein concentration of Mo¨ssbauer samples and used to estimate the amount of oxidized [4Fe–4S] cluster loss with irradiation time. The obtained values, presented in Table 2, agree with the ones obtained from Mo¨ssbauer analysis confirming, once more, the conversion of the iron–sulfur center into a different iron species. Therefore, combining the Mo¨ssbauer and UV–visible data, we propose that during the initial 45 min of irradiation, the [4Fe–4S]2? cluster is converted into a [2Fe–2S]2?

Table 1 Mo¨ssbauer percentages determined for the [4Fe–4S] cluster and species I and II with irradiation time Speciesa

Irradiation time 0 min

[4Fe–4S]

20 min

45 min

80 min

120 min

100

82

72

57

31

I

0

18

28

31

12

II

0

0

0

12

57

a

Species observed in the Mo¨ssbauer samples

Table 2 Mo¨ssbauer and UV–visible percentages attributed to the [4Fe–4S]2? cluster %a

Irradiation time 0 min

20 min

45 min

80 min

120 min

Mo¨ssbauer

100

82

72

57

31

UV–visible, 410 nm

100

84

78

65

33

Samples for Mo¨ssbauer and UV–visible spectroscopies were similar in terms of preparation and final concentration a Percentages calculated from the Mo¨ssbauer and UV–visible analysis

form. Longer exposure times seem to degrade both the [4Fe–4S]2? and the [2Fe–2S]2? states producing monomeric high-spin ferric ions that aggregate to produce oxo/ hidroxo-ferric polynuclear aggregates species (Muh et al. 1997). Electrophoretic mobility shift assay To study the DNA-binding ability of UV-irradiated rEndoIII, electrophoretic mobility shift assays (EMSA) were performed. The aim of the study was to evaluate the effect of UV-C irradiation on the affinity of EndoIII to DNA. Therefore, exposed and non-exposed proteins were incubated with supercoiled plasmid DNA, pUC18, and complex formation was analyzed by agarose gel electrophoresis. The relative EndoIII-pDNA complex fraction was plotted as a function of protein concentration and fitted to a Hill equation (Timoteo et al. 2012) (Supplementary Fig. S2b). When native (non-irradiated) protein was used, we were able to verify a protein-pDNA complex dependent on protein concentration. In our experimental conditions, an apparent dissociation constant, Kd, was determined to be equal to 0.85 ± 0.04 and n = 1.63 ± 0.08. At a proteinDNA ratio of 500 (5 lM of protein), practically all freeDNA band was shifted to the complex form (lane 14 on the gel in Supplementary Fig. S2a). Pre-irradiation of the protein prior to DNA binding affects the DNA-binding function of the protein (Fig. 5). The affinity of the protein for plasmid DNA decreased with the irradiation time when exposed until

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Fig. 5 DNA binding of UV-irradiated EndoIII assessed by EMSA in 100 mM Tris–HCl, pH 7.1. Binding of a native, b–f 30 min (197 mJ), 45 (296 mJ), 60 (394 mJ), 120 min (788 mJ) and 6 h (2,365 mJ) irradiated E. coli rEndoIII to supercoiled plasmid DNA. In each gel lane 1, 1-kB ladder; lane 2, supercoiled pUC18 (10 nM); lane 3, rEndoIII (10 lM); lanes 4–16, binding of rEndoIII (0.04, 0.08, 0.17,

0.25, 0.33, 0.67, 1.00, 1.34, 1.68, 2.51, 5.00, 7.50, 10 lM) to 10 nM of pUC18. The electrophoretic mobilities were assessed by 1 % agarose gel electrophoresis in 19 TAE buffer. The gel was stained with SYBRSafe. The free supercoiled DNA band (SC) and proteinDNA band (Complex) are indicated on the right of the gel

30 min (Fig. 5b). In these conditions, the Hill fits showed an increase on the Kd value from 0.85 (for the non-irradiated protein) to 2.49 (30 min-irradiation). In EMSAs using rEndoIII irradiated for longer times (from 45 to 60 min, Fig. 5c, d), protein-DNA complexes with higher molecular masses were observed (with higher molecular masses than the shifted band on the nonirradiated protein—Fig. 5a), some of them not migrating into the gel. These larger complexes were formed at lower protein-DNA ratios (when compared with the non-irradiated protein—Fig. 5a); nevertheless, fewer protein molecules bound DNA molecules, since less free-DNA was shifted. Moreover, these larger proteinDNA complexes seem to be unstable, thus smearing on the gel. Irradiation for 2 h or longer times (Fig. 5e, f) resulted in the inactivation of the binding function of rEndoIII, as no shifting of the free DNA band was observed.

Discussion

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DNA damage caused by UV-C radiation has been largely described in the literature (Silva et al. 2012; Zhang et al. 1997; Wei et al. 1997; Ramirez-Guadiana et al. 2012; Pfeifer et al. 2005). Therefore, our objective was to identify the primary target of this type of radiation in a protein that plays a central role in DNA repair, EndoIII, as well as its influence in the protein’s ability to bind DNA. In theory, an UV-C source, such as the one used in this work (emission band at 254 nm), should not have a stronger direct effect on proteins than it should on DNA since the first ones should not absorb much at this wavelength while, on the other hand, DNA (with maximum of absorption near 260 nm) should be the one to absorb more energy, being a better candidate to suffer direct damage from exposure to this type of radiation. However, this type of radiation can also cause damage by forming reactive oxygen species

Radiat Environ Biophys

such as 1O2 (Bruge et al. 2003; Zhang et al. 1997; Wei et al. 1997) that may trigger a number of damaging reactions. Such effects are denominated indirect effects since damage is caused not by the direct effect of the radiation itself. The experimental observations pointed to the fact that ROS cause damage to other biomolecules than DNA, namely to proteins involved in DNA repair, affecting their functions, eventually culminating in cell death. A model of cell death by protein damage in irradiated cells was recently proposed by Michael J. Daly, which states that, in radiation-sensitive cells, proteins are the primary target of ROS generated by radiolysis of water (Daly 2012). Cell death seems to result from the progressive accumulation of oxidative damage of the proteome, in particular due to oxidation of iron-containing proteins, resulting in the inactivation of vital physiological catalytic function (Slade and Radman 2011). As far as we know, the effect of radiation on DNA repair enzymes has not been reported, specifically its influence on the iron–sulfur cluster of the protein. Is the iron–sulfur center converted into another iron species or is it immediately released from the polypeptide chain? To test this hypothesis, we applied different spectroscopic and biochemical methods in order to identify structural modifications on the E. coli DNA repair enzyme endonuclease III promoted by UV-C radiation exposure and their effects on the DNA-binding activity of the protein. If protein exposure to UV-C, or other potential source of ROS or oxidative stress inducing species, leads to the consequences obtained in this work, it was expected to observe an effect on rEndoIII ability to bind DNA. This was verified when electrophoretic mobility shift assays were performed with non-exposed and exposed samples incubated with plasmid DNA. In the experimental conditions tested, native non-irradiated EndoIII-DNA complex has a Kd of about 0.85 lM, a value similar to other DNAbinding proteins (Swinger and Rice 2004; Timoteo et al. 2012; Castruita et al. 2006). When the protein was preirradiated before incubation with DNA, with exposure times up to 30 min, the Kd increases indicating a smaller binding affinity caused by irradiation. When the protein was exposed to UV-C radiation for 2 or 6 h, in the experimental conditions tested, it lost its DNA-binding ability becoming a non-functional enzyme. If one compares these observations with the spectroscopic data, lower affinity to DNA can be explained by structural modification with the loss of secondary and tertiary structural features of the protein, [4Fe–4S]2? cluster conversion and dimerization through covalent dityrosine cross-links. The conversion of a [4Fe–4S]2? cluster into a [3Fe– 4S]1? or a [2Fe–2S]2? cluster is already known from the literature (Agarwalla et al. 2004; Cunningham et al. 1989).

However, the radiation-induced conversion of a [4Fe– 4S]2? cluster protein into a [2Fe–2S]2? cluster containing one is proposed here for the first time. One example also related to oxidative stress effects promoting such type of iron–sulfur interconversion was first observed in the fumarate and nitrate reduction (FNR) regulatory proteins, oxygen sensing transcription factors that control the switch between aerobic and anaerobic metabolism in prokaryotes. Under anaerobic conditions [4Fe–4S]2? containing FNR is a homodimer that binds to DNA; but the presence of high levels of O2 converts the [4Fe–4S]2? into a [2Fe–2S]2? form and shifts the dimer to a monomer non-active protein form (Jervis et al. 2009; Crack et al. 2008a). In another study, Barton and collaborators (Romano et al. 2011) proposed that, besides the [4Fe–4S] cluster, some aromatic residues also play a key role in the charge transport between EndoIII and DNA. Analysis of the tridimensional structure of E. coli EndoIII reveals that one of the residues mentioned in the paper, Y82, is very exposed and, therefore, a very good candidate to establish dityrosine cross-links. On the other hand, the authors also pointed that other two residues, W178 and Y185, that should be relevant for the stability of the [4Fe–4S] cluster apparently did not produce any structural modification and, contrary to what was expected by the authors, resulted in an increase in the protein/DNA coupling. We believe that this behavior support our findings, as it also demonstrates that not only the [4Fe–4S] cluster but also the protein structure itself can be important to the protein–DNA interaction and, consequently, to its function toward DNA repair. Although there is no evidence that the EndoIII sensitivity to the radiation-induced oxidative stress demonstrated in this work can act as a regulatory mechanism inside a cell, one cannot ignore that, at a molecular level, the damage caused by sources of radiation is not restricted to DNA molecules but also to enzyme molecules, in particular to iron–sulfur-containing proteins involved in DNA repair systems. Rationalization of these results in cellular context strengthens the argument that cellular sensitivity to radiation can be due to loss of radiation-induced damage repair ability. Acknowledgments This research was supported by Fundac¸a˜o para a Cieˆncia e Tecnologia, Ministe´rio da Educac¸a˜o e Cieˆncia, grant PTDC/SAU-SAP/111482/2009 (P.T.), PTDC/QUI/64248/2006 (to A.S.P), grant SFRH/BPD/48430/2008, a Post-Doctoral grant to F.F. Requimte was funded in part by grant PEst-C/EQB/LA0006/2011 from FCT/MEC.

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