DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae

DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in... Abstract Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS. Tpa1, AlkBH3, AlkB, dioxygenase, DNA alkylation, DNA repair, iron ABBREVIATIONS 2OG 2-oxoglutarate N1-meA N1-methyl adenine N3-meC N3-Methyl-Cytosine MMS Methyl methane sulfonate MNNG Methyl-3-nitro-1-nitrosoguanidine YNB Yeast Nitrogen Base INTRODUCTION Iron-responsive transcription factor Aft1 (activators of ferrous transport) and its paralog Aft2 regulate gene expression in response to low level of iron in Saccharomyces cerevisiae (Yamaguchi-Iwai, Dancis and Klausner 1995; Yamaguchi-Iwai et al. 1996; Blaiseau, Lesuisse and Camadro 2001). Depletion of cellular iron causes Aft1/Aft2 to enter nucleus and upregulate transcription of genes involved in the iron uptake (Chen et al. 2004). When cellular iron level is restored, Aft1/Aft2 dissociates from DNA and exported to cytoplasm, resulting in switching off transcription of the target genes (Yamaguchi-Iwai et al. 2002; Ojeda et al. 2006; Ueta et al. 2007; Poor et al. 2014). Aft1/Aft2 recognize similar promoter element and functionally compensate each other (Rutherford, Jaron and Winge 2003). Genetic studies revealed that aft1Δ mutant cells exhibit poor growth under low-iron conditions (Yamaguchi-Iwai, Dancis and Klausner 1995), aft2Δ mutant cells show no growth defects under these conditions (Rutherford, Jaron and Winge 2003). However, consistent with the overlapping function of Aft1 and Aft2, an aft1Δaft2Δ double mutant is more sensitive to low-iron growth conditions than a single aft1Δ mutant alone (Blaiseau, Lesuisse and Camadro 2001). Due to their role in iron homeostasis in yeast, Aft1/Aft2 also indirectly influence a diverse range of cellular processes, including cell wall stability, protein transport, mitochondrial function and DNA damage response (Shakoury-Elizeh et al. 2004; Berthelet et al. 2010; Hamza and Baetz 2012). Role of iron uptake in protecting the cells from DNA damaging chemicals were revealed by studying aft1 deletion mutant. Mutant aft1Δ cells are known to be hypersensitive to DNA damaging agents such as cisplatin, hydroxyurea and methylating agents methyl methane sulfonate (MMS) (Lee et al. 2005; Dubacq et al. 2006; Kimura, Ohashi and Naganuma 2007). Although this observation suggests that iron might have some protective effects against DNA alkylation damage but the mechanism remains poorly understood. MMS exposure results in several cytotoxic adducts in genomic DNA including, N1-methyladenine (N1-MeA) and N3-methylcytosine (N3-MeC) (Wyatt and Pittman 2006). Most organisms employ the AlkB family of enzymes for direct reversal of these adducts (Fedeles et al. 2015). Escherichia coli AlkB is a bona fide oxidative dealkylation DNA repair enzyme that protects the bacterial genome against alkylation damage (Falnes, Johansen and Seeberg 2002; Trewick et al. 2002). AlkB uses molecular oxygen to oxidize the methyl groups on N1-MeA and N3-MeC; the oxidized methyl group is subsequently released as formaldehyde, regenerating the adenine and cytosine, respectively (Shen et al. 2014). Most eukaryotes have AlkB homologs, including budding yeast S. cerevisiae. AlkB homolog in S. cerevisiae is known as Tpa1 (transcription and polyadenylation associated), and ectopic expression of TPA1 rescues AlkB deficiency in E. coli (Shivange et al. 2014). Mammalian cells have several AlkB homologs but ALKBH2 and ALKBH3 codes for functional nuclear DNA repair enzymes and protect cells from DNA damage following acute inflammation (Aas et al. 2003; Calvo et al. 2012). The AlkB enzyme belongs to non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases superfamily. These enzymes catalyze broad range of oxidation reactions (Fedeles et al. 2015). For example, Tet1–3 (Ten-eleven translocation) proteins reverse epigenetic modification 5mC to 5-hydroxymethyl-cytosine and further to 5-formyl-cytosine and 5-carboxyl-cytosine (Tahiliani et al. 2009; Ito et al. 2010; He et al. 2011; Ito et al. 2011; Pfaffeneder et al. 2011); AlkB homolog 5 (ALKBH5) and FTO (Jia et al. 2008; Lando et al. 2012; Zheng et al. 2013; Zhang et al. 2016) remove N6-methyladenosine (N6-meA). Besides nucleic acid modification, Fe(II)/2OG-dependent dioxygenases carry out variety of cellular functions, including post-translational modification of collagen proteins by prolyl hydroxylases (Ricard-Blum 2011); synthesis of quinolinic acid from 3-hydroxyanthranilic acid in the tryptophan-NAD+ biosynthetic pathway by metabolic enzyme 3-hydroxy-anthranilate dioxygenase (Brkic, Kovacevic and Tomic 2015); oxidative modification of the nuclear factor κB transcription factor by nuclear protein Pirin acts as (Liu et al. 2013), and so on. Structurally these proteins are characterized by eight antiparallel β-strands forming two β-sheets that commonly referred to as jelly-roll motif. Between the two β-sheets, an Fe(II) atom is bound via three conserved residues: a histidine, an aspartate/glutamate and a histidine (HXD/E…H). These enzymes require Fe2+, 2OG, O2 and ascorbate. AlkB family of Fe(II)/2OG-dependent dioxygenases are known to be regulated by availability of cofactors, including 2OG, oxygen and iron. For example, Tet enzymes, AlkB human homologs and histone demethylases are susceptible to inhibition by 2-hydroxyglutarate (2HG), a metabolite which is structurally similar to 2OG and accumulates in tumor cells due to mutations in isocitrate dehydrogenase (Xu et al. 2011; Wang et al. 2015). AlkB family of enzymes is also inhibited by lack of oxygen during cellular hypoxia (Anindya 2017). Although it has been speculated that availability of iron might influence the activity of Fe(II)/2OG-dependent dioxygenases family of enzymes (Mole 2010; Salminen, Kauppinen and Kaarniranta 2015), but to our knowledge no experimental or clinical studies exist to support this. In this paper, we examined how the cellular iron level impacts the function of Fe(II)/2OG-dependent dioxygenases family of enzymes involved in the DNA repair. We studied bacterial AlkB, S. cerevisiae AlkB homolog Tpa1 and human AlkB homolog AlkBH3, which are involved in alkylated DNA repair. Using previously described genetic systems we show that maintenance of iron homeostasis by transcription factor Aft1/Aft2 is crucial for the function under iron depletion condition. Our results suggest that function of Fe(II)/2OG-dependent dioxygenases family of enzymes will be affected if cellular iron level is not maintained in response to environmental conditions. MATERIALS AND METHODS Yeast strains and media Saccharomyces cerevisiae W3031a and its isogenic strains used in this study are described in Table 1. All knockout mutants were generated by using the one-step PCR method. Auxotrophic marker genes HIS3, TRP1, URA3 and LEU2 were amplified from pRS300 series vectors by using specific primers. Cells were grown in YPD or synthetic minimal media. To integrate the functional genes into yeast genome, counterselection method was used by selecting ura3 mutant cells on minimal media containing 5-fluoroorotic acid. Table 1. Saccharomyces cerevisiae strains used in this study. S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  View Large Table 1. Saccharomyces cerevisiae strains used in this study. S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  View Large Spot assay SD minimal medium was prepared by adding the appropriate amino acids to Yeast Nitrogen Base (YNB) without amino acids and supplemented with 2% glucose. This medium contains 0.2 mg/L iron from YNB; therefore, different concentrations of ferrozine (1–2 mM) were added for iron depletion. Approximately 6 × 106 cells were taken from exponentially growing culture, washed and suspended in 1 ml of potassium phosphate buffer pH 7.4. Seven microliters of 10-fold serially diluted four dilutions were spotted onto minimal media containing different concentrations of DNA damaging agents MMS or MNNG that supplemented without or with 1 mM ferrozine. Growth was monitored at 30°C for 5 days. Survival assay Cells were grown at 30˚C for overnight from a single colony in SD media containing 1 mM ferrozine. Thereafter, cells were treated with different concentrations of MMS. Approximately 4 × 103 cells were spread onto YPD medium and grown at 30˚C for 72 h. Cell survival was monitored by the number of colonies that were present. The number of colonies from untreated cells was taken as 100% and the percentage of cell viability was plotted by using Graph Pad-Prism software. RESULTS AND DISCUSSION Iron deficiency leads to inactivation of Tpa1 function Saccharomyces cerevisiae AFT1 gene is required for growth in iron-depleted media (Yamaguchi-Iwai, Dancis and Klausner 1995). As a first step in our analysis of growth under iron-limiting and iron-depleted condition, we examined growth of aft1Δ, aft2Δ and aft1Δ aft2Δ strains. We have used SD minimal medium, which contains low level of iron (0.2 mg/L). We have used the increasing concentration of iron chelator ferrozine (1–2 mM) to deplete iron and monitored growth of these strains by spot dilution assays on SD agar plates. As shown in Fig. 1A, aft1Δ, aft2Δ and aft1Δ aft2Δ strains showed cell growth comparable to wild-type when ferrozine concentration was between 1 and 1.5 mM, suggesting limited availability of iron in the media. In this study, we refer this experimental condition as iron-limiting. However, when ferrozine concentration was increased to 1.75 mM and above, aft1Δ aft2Δ strains showed growth defect, suggesting severe iron depletion under these conditions. Single-mutant strains aft1Δ, aft2Δ did not show any sensitivity to iron depletion under present experimental condition suggesting overlapping function of Aft1 and Aft2, as also observed previously (Blaiseau, Lesuisse and Camadro 2001). Figure 1. View largeDownload slide Deletion of genes encoding Aft1 and Aft2 lead to MMS sensitivity in an tpa1mag1Δ background. (A) Mild growth defect phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), aft1Δ, aft2Δ and aft11Δaft2Δ strains were spotted on SD supplemented with increasing concentrations of iron chelator ferrozine to deplete iron. The plates are shown after 4 days of incubation at 30°C. (B) Normal growth phenotype of aft11Δaft2Δ in the presence of MMS. Wild-type strain (W303), aft1Δ, aft2Δ, aft11Δaft2Δ and tpa1Δ strains were spotted on SD media containing DNA alkylating agent MMS as before. (C) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of ferrozine. Wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, mag1Δaft1Δ and mag1Δaft1Δaft2Δ strains were spotted on SD media containing either MMS (middle) or iron chelator ferrozine (left) or both (right) as before. (D) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of iron chelator curcumin. Wild-type and mutant strains were spotted on SD media containing MMS and iron chelator curcumin as before. (E) Normal growth phenotype of wild-type and mutant strains in the presence of ferrozine and SN1 alkylating agent MNNG. Strains were spotted on SD media containing either DNA alkylating agent MNNG (middle) or iron chelator ferrozine (left panel) or both (right panel). Figure 1. View largeDownload slide Deletion of genes encoding Aft1 and Aft2 lead to MMS sensitivity in an tpa1mag1Δ background. (A) Mild growth defect phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), aft1Δ, aft2Δ and aft11Δaft2Δ strains were spotted on SD supplemented with increasing concentrations of iron chelator ferrozine to deplete iron. The plates are shown after 4 days of incubation at 30°C. (B) Normal growth phenotype of aft11Δaft2Δ in the presence of MMS. Wild-type strain (W303), aft1Δ, aft2Δ, aft11Δaft2Δ and tpa1Δ strains were spotted on SD media containing DNA alkylating agent MMS as before. (C) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of ferrozine. Wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, mag1Δaft1Δ and mag1Δaft1Δaft2Δ strains were spotted on SD media containing either MMS (middle) or iron chelator ferrozine (left) or both (right) as before. (D) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of iron chelator curcumin. Wild-type and mutant strains were spotted on SD media containing MMS and iron chelator curcumin as before. (E) Normal growth phenotype of wild-type and mutant strains in the presence of ferrozine and SN1 alkylating agent MNNG. Strains were spotted on SD media containing either DNA alkylating agent MNNG (middle) or iron chelator ferrozine (left panel) or both (right panel). Having established a condition where iron level is limiting but not totally depleted, we wanted to examine the role of iron-responsive transcription factor Aft1/Aft2 on the activity of Fe(II)/2OG-dependent dioxygenases. We hypothesized that under iron-limiting condition, Fe(II)/2OG-dependent dioxygenases involved in alkylated-DNA repair might exhibit reduced activity causing MMS sensitivity. In S. cerevisiae, MMS-induced DNA damage is repaired synergistically by the two different pathways, namely direct repair by Fe(II)/2OG-dependent dioxygenase Tpa1 and BER DNA glycosylase Mag1 (Shivange et al. 2014). It was also observed previously that the tpa1Δ deletion mutant had little MMS sensitivity at low concentration of MMS, due to Mag1 activity. Similarly, mag1Δ deletion also caused only modest MMS sensitivity. However, the double mutant tpa1Δ mag1Δ was hypersensitive to MMS (Shivange et al. 2014). We reasoned that under iron-limiting condition and in the absence of Aft1/Aft2, mag1Δ mutant cells would display MMS hypersensitivity akin to tpa1Δ mag1Δ cells, due to functionally defective Tpa1. Therefore, we generated aft1Δ, aft2Δ and aft1Δaft2Δ deletion strains and compared their survival with tpa1Δ strain at low concentration of MMS (0.0075%) by spot dilution assays on SD agar plates. As shown in Fig. 1B, none of these mutants were sensitive to low concentration of MMS used, suggesting that Aft1/Aft2 had no iron-independent and direct mechanism of causing MMS sensitivity. Having confirmed that aft1Δ, aft2Δ and aft1Δaft2Δ deletion strains being not sensitive to low dose of MMS, we made aft1Δmag1Δ and aft2Δmag1Δ double mutant and aft1Δaft2Δmag1Δ triple mutant strains and compared MMS sensitivity with tpa1Δmag1Δ strain under different conditions (Fig. 1C). As expected, wild-type cells grew in the presence of 0.0075% MMS, whereas mag1Δtpa1Δ mutants cells were extremely sensitive, in agreement with previous result (Shivange et al. 2014). Notably, aft1Δaft2Δmag1Δ showed stronger MMS sensitivity compared to aft1Δmag1Δ and aft2Δmag1Δ double mutant when cells were gown in the presence of 1 mM ferrozine. This growth defect is due to MMS exposure as without MMS, aft1Δaft2Δ mutant strain exhibited normal growth (Fig. 1C, left panel). Another iron chelator that is known to suppress the growth of S. cerevisiae is curcumin, a yellow pigment found in Indian herb turmeric (Minear et al. 2011). We performed growth assay with different concentrations of curcumin and found that at 50 μM concentration of curcumin, growth of wild-type and mutant yeast cells was not affected (Fig. 1D). The concentration of curcumin used here was significantly lower than what was reported to deplete cellular iron level (Azad et al. 2013) and thus could be considered iron-limiting. Next we investigated the effect of low concentration of MMS in the presence of curcumin. We observed that wild-type cells grew in the presence of 0.0075% MMS and 50 μM curcumin, whereas mag1Δtpa1Δ mutant cells were extremely sensitive, as expected. Interestingly, aft1Δaft2Δmag1Δ showed MMS sensitivity akin to mag1Δtpa1Δ mutants and mag1Δ single mutant was much less sensitive. These data support our hypothesis that when iron is limiting, iron-responsive transcription factors are needed to maintain cellular iron level and thereby keeping the Fe(II)/2OG-dependent dioxygenase Tpa1 active. Lack of iron homeostasis, however, rendered Tpa1 inactive under the condition where iron was limiting. It was reported earlier that AlkB family members repair only the N-alkylation damage caused by SN-2 type of alkylating agent MMS, but not involved in repair of the O-alkylation adducts such as O6-meG caused by SN-1 type of alkylating agent 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG). In order to examine if iron depletion leads to sensitivity to O-alkylating agents, we analyzed cell survival in the presence of MNNG by spot dilution method on SD agar plates. As shown in Fig. 1D, both aft1Δaft2Δmag1Δ and tpa1Δmag1Δ strains could tolerate 15% of MNNG due to active DNA repair. As Tpa1 and Mag1 are not involved in repairing MNNG-induced damage, double mutant tpa1Δamag1Δ strain did not show any sensitivity to MNNG compared to wild type (Fig. 1E). This result confirms that MMS hypersensitivity of aft1Δaft2Δmag1Δ cells could be due to lack of Tpa1-mediated repair under iron-limiting condition. Iron deficiency leads to inactivation of bacterial AlkB and human AlkBH3 function Saccharomyces cerevisiae Fe(II)/2OG-dependent dioxygenase Tpa1 is a functional ortholog of bacterial AlkB. Although there are nine AlkB family proteins in human, AlkBH2 and AlkBH3 are bona fide nuclear DNA repair enzymes. We hypothesized that when iron depletion could inhibit Tpa1 DNA repair activity, it would also similarly affect bacterial AlkB or human AlkB orthologs. Therefore, we decided to study two more Fe(II)/2OG-dependent dioxygenases, namely E. coli AlkB and human AlkBH3 in S. cerevisiae under iron-limiting condition. We used a plasmid shuffle strategy to introduce the bacterial or human gene variant in the tpa1Δ mutants. Once we obtained the complementation strains, we wanted to test whether AlkB or AlkBH3 could rescue MMS hypersensitivity of mag1Δtpa1Δ strain and rescue the effect of deletion of Tpa1 on MMS sensitivity. For this, we performed spot dilution assays on SD agar plates containing a range of MMS concentrations from 0.0075% to 0.015%. Figure 2A shows that the tpa1Δmag1Δ strain is exquisitely sensitive to MMS, but when ALKB and ALKBH3 complemented TPA1, the strains were able to rescue the sensitivity to MMS. This restoration of resistance to MMS by AlkB and AlkBH3 suggests functional complementation. Next, we examined the effect of limiting iron concentration on the activity of AlkB and AlkBH3. For this, we introduced E. coli AlkB and human AlkBH3 in aft1Δaft2Δmag1Δtpa1Δ background. As shown in Fig. 2B, AlkB and AlkBH3 expressing cells showed strong MMS sensitivity of when cells were gown in the presence of 1 mM ferrozine. This growth defect is further pronounced in aft1Δaft2Δ genetic background. This increase in sensitivity to MMS is similar to that observed for aft1Δaft2Δmag1Δ strain. This result supports our hypothesis that lack of Aft1/Aft2 in the iron-limiting condition severely affects the activity of AlkB family of Fe(II)/2OG-dependent dioxygenases and results in MMS sensitivity. Figure 2. View largeDownload slide Functional complementation of S. cerevisiae Tpa1 by E. coli AlkB and human AlkBH3. (A) MMS sensitivity phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were spotted on SD media containing MMS. The plates are shown after 4 days of incubation at 30°C. (B) Hypersensitivity to alkylating agent MMS in the absence of genes coding Aft1 and Aft2. Wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were grown as before in the presence of MMS and iron chelator ferrozine. Figure 2. View largeDownload slide Functional complementation of S. cerevisiae Tpa1 by E. coli AlkB and human AlkBH3. (A) MMS sensitivity phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were spotted on SD media containing MMS. The plates are shown after 4 days of incubation at 30°C. (B) Hypersensitivity to alkylating agent MMS in the absence of genes coding Aft1 and Aft2. Wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were grown as before in the presence of MMS and iron chelator ferrozine. Iron deficiency results in reduced cell survival after exposure to MMS We quantified the ability of wild-type and mutant strains to form colonies following acute exposure to MMS. We treated the cells in SD media containing 0.005%, 0.015% and 0.030% MMS and 1 mM ferrozine and plated a fixed number of cells of each strain on YPD agar plates without MMS. The surviving colonies were counted and expressed as percent survival by comparing with untreated cells (Fig. 3A and B). There was no survival of aft1Δaft2Δmag1Δ and mag1Δtpa1Δ strains observed in SD medium supplemented with ferrozine and MMS. This result is in agreement with our earlier observation and suggests that Tpa1 function is affected under chronic iron-limiting condition and affects cell survival to MMS exposure. We have also quantified the ability of AlkB and AlkBH3 expressing strains to survive and colonies after acute exposure to MMS. We treated each of the strains in SD media containing 0.005%, 0.015% and 0.030% MMS and 1 mM ferrozine and plated a fixed number of cells on YPD agar plates without MMS. The surviving colonies were counted in each plate and expressed as percent survival by comparing to untreated cells (Fig. 3C). Strains expressing AlkB and AlkBH3 in aft1Δaft2Δ background were failed to grow similar to mag1Δtpa1Δ strains in SD medium supplemented with ferrozine and MMS (Fig. 3D). This result is comparable to Tpa1 expressing cells and suggests that function of AlkB and AlkBH3 is equally affected under chronic iron-limiting condition in the absence of iron homeostasis by Aft1/Aft2. Figure 3. View largeDownload slide Survival analysis of strains expressing S. cerevisiae Tpa1 or E.coli AlkB or human AlkBH3. (A) Survival curve of wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, aft1Δaft2Δ and aft11Δaft2Δmag1Δ strains grown in cultures containing 0.05%, 0.15% and 0.30% (v/v) of methylating agent MMS. Survival was determined by plating cells on LB agar at 37°C for 20 h and colony counting. The viability of cells not treated with MMS was 100%. Error bars indicate the standard error of the mean. All the strains are the isogenic derivatives of W303. (B) Assessment of MMS sensitivity of tpa1Δmag1Δ and aft11Δaft2Δmag1Δ strains. Colonies of each strain were streaked on an SD plate containing iron chelator ferrozine and either no MMS or 0.0075% (v/v) MMS. As a control, wild-type strain (W303) was streaked. Plates were incubated at 30°C for 4 days and photographed. (C) Survival curve of strains expressing E. coli AlkB and human AlkBH3. Cell viability was determined as before. (D) Assessment of MMS sensitivity of E. coli AlkB or human AlkBH3 in aft1Δaft2Δ background. All treatments were as described in B. Error bars represent the SEM of six replicates from three representative experiments that were repeated two times. *P < 0.05, ANOVA. Figure 3. View largeDownload slide Survival analysis of strains expressing S. cerevisiae Tpa1 or E.coli AlkB or human AlkBH3. (A) Survival curve of wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, aft1Δaft2Δ and aft11Δaft2Δmag1Δ strains grown in cultures containing 0.05%, 0.15% and 0.30% (v/v) of methylating agent MMS. Survival was determined by plating cells on LB agar at 37°C for 20 h and colony counting. The viability of cells not treated with MMS was 100%. Error bars indicate the standard error of the mean. All the strains are the isogenic derivatives of W303. (B) Assessment of MMS sensitivity of tpa1Δmag1Δ and aft11Δaft2Δmag1Δ strains. Colonies of each strain were streaked on an SD plate containing iron chelator ferrozine and either no MMS or 0.0075% (v/v) MMS. As a control, wild-type strain (W303) was streaked. Plates were incubated at 30°C for 4 days and photographed. (C) Survival curve of strains expressing E. coli AlkB and human AlkBH3. Cell viability was determined as before. (D) Assessment of MMS sensitivity of E. coli AlkB or human AlkBH3 in aft1Δaft2Δ background. All treatments were as described in B. Error bars represent the SEM of six replicates from three representative experiments that were repeated two times. *P < 0.05, ANOVA. It was also published before that when S. cerevisiae cells are grown under iron-depleted condition, mRNA levels of several proteins containing Fe-S cluster proteins, such as aconitase and succinate dehydrogenase, are downregulated. Although Fe(II)/2OG-dependent dioxygenases like Tpa1 are iron-containing enzymes, they bind non-heme Fe(II) and not the Fe-S cluster. Furthermore, multiple microarray studies carried out to determine the genes regulated by iron levels revealed that mRNA level of Tpa1 is not downregulated under low iron conditions (Shakoury-Elizeh et al. 2004; Berthelet et al. 2010). TPA1 promoter does not have recognition sequence for Aft1/Aft2 nor were they reported to be part of ‘iron regulon’ genes (Philpott et al. 2002; Philpott and Protchenko 2008). Therefore, our data presented here indicate that in the absence of Aft1/Aft2, lack of iron homeostasis is most likely responsible for MMS hypersensitivity of aft1Δaft2Δmag1Δ strain. In the absence of iron responsive transcription factor Aft1, S. cerevisiae is sensitive to high dose of MMS (0.05%) when grown in completely iron-depleted media (Berthelet et al. 2010). This MMS sensitivity was restored by exogenously supplying iron. It remains completely unknown how Aft1 might influence MMS sensitivity. Taken together, our data suggest that depletion of iron renders Fe(II)/2OG-dependent dioxygenases inactive and support the hypothesis that cellular iron concentration could be an important regulator of AlkB family of enzymes. Limited bioavailability has been shown to affect Fe(II)/2OG-dependent dioxygenases family of enzyme prolyl-hydroxylase involved in the degradation of hypoxia inducible factor (Knowles et al. 2006; Pan et al. 2007). Indeed, individuals with severe iron-deficient anemia were reported to have impaired hypoxic response (Frise et al. 2016). Our results suggest that function of Fe(II)/2OG-dependent dioxygenases family of DNA repair enzymes will be affected if cellular iron level is not maintained in response to environmental conditions. Because iron deficiency affects vast number individuals worldwide, DNA repair defect due to iron deficiency could be of considerable importance for human health and disease. Acknowledgements Authors acknowledge technical help from Monisha Mohan and Richa Nigam. FUNDING The work was funded by Extra-mural research project (EMR/2016/0 05135) funded by Science and Engineering Research Board (SERB), Government of India. Conflict of interest. None declared. REFERENCES Aas PA, Otterlei M, Falnes PO et al.   Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature  2003; 421: 859– 63. Google Scholar CrossRef Search ADS PubMed  Anindya R. Non-heme dioxygenases in tumor hypoxia: they’re all bound with the same fate. DNA Repair  2017; 49: 21– 5. Google Scholar CrossRef Search ADS PubMed  Azad GK, Singh V, Golla U et al.   Depletion of cellular iron by curcumin leads to alteration in histone acetylation and degradation of Sml1p in Saccharomyces cerevisiae. PLoS One  2013; 8: e59003. 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DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae

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Abstract

Abstract Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS. Tpa1, AlkBH3, AlkB, dioxygenase, DNA alkylation, DNA repair, iron ABBREVIATIONS 2OG 2-oxoglutarate N1-meA N1-methyl adenine N3-meC N3-Methyl-Cytosine MMS Methyl methane sulfonate MNNG Methyl-3-nitro-1-nitrosoguanidine YNB Yeast Nitrogen Base INTRODUCTION Iron-responsive transcription factor Aft1 (activators of ferrous transport) and its paralog Aft2 regulate gene expression in response to low level of iron in Saccharomyces cerevisiae (Yamaguchi-Iwai, Dancis and Klausner 1995; Yamaguchi-Iwai et al. 1996; Blaiseau, Lesuisse and Camadro 2001). Depletion of cellular iron causes Aft1/Aft2 to enter nucleus and upregulate transcription of genes involved in the iron uptake (Chen et al. 2004). When cellular iron level is restored, Aft1/Aft2 dissociates from DNA and exported to cytoplasm, resulting in switching off transcription of the target genes (Yamaguchi-Iwai et al. 2002; Ojeda et al. 2006; Ueta et al. 2007; Poor et al. 2014). Aft1/Aft2 recognize similar promoter element and functionally compensate each other (Rutherford, Jaron and Winge 2003). Genetic studies revealed that aft1Δ mutant cells exhibit poor growth under low-iron conditions (Yamaguchi-Iwai, Dancis and Klausner 1995), aft2Δ mutant cells show no growth defects under these conditions (Rutherford, Jaron and Winge 2003). However, consistent with the overlapping function of Aft1 and Aft2, an aft1Δaft2Δ double mutant is more sensitive to low-iron growth conditions than a single aft1Δ mutant alone (Blaiseau, Lesuisse and Camadro 2001). Due to their role in iron homeostasis in yeast, Aft1/Aft2 also indirectly influence a diverse range of cellular processes, including cell wall stability, protein transport, mitochondrial function and DNA damage response (Shakoury-Elizeh et al. 2004; Berthelet et al. 2010; Hamza and Baetz 2012). Role of iron uptake in protecting the cells from DNA damaging chemicals were revealed by studying aft1 deletion mutant. Mutant aft1Δ cells are known to be hypersensitive to DNA damaging agents such as cisplatin, hydroxyurea and methylating agents methyl methane sulfonate (MMS) (Lee et al. 2005; Dubacq et al. 2006; Kimura, Ohashi and Naganuma 2007). Although this observation suggests that iron might have some protective effects against DNA alkylation damage but the mechanism remains poorly understood. MMS exposure results in several cytotoxic adducts in genomic DNA including, N1-methyladenine (N1-MeA) and N3-methylcytosine (N3-MeC) (Wyatt and Pittman 2006). Most organisms employ the AlkB family of enzymes for direct reversal of these adducts (Fedeles et al. 2015). Escherichia coli AlkB is a bona fide oxidative dealkylation DNA repair enzyme that protects the bacterial genome against alkylation damage (Falnes, Johansen and Seeberg 2002; Trewick et al. 2002). AlkB uses molecular oxygen to oxidize the methyl groups on N1-MeA and N3-MeC; the oxidized methyl group is subsequently released as formaldehyde, regenerating the adenine and cytosine, respectively (Shen et al. 2014). Most eukaryotes have AlkB homologs, including budding yeast S. cerevisiae. AlkB homolog in S. cerevisiae is known as Tpa1 (transcription and polyadenylation associated), and ectopic expression of TPA1 rescues AlkB deficiency in E. coli (Shivange et al. 2014). Mammalian cells have several AlkB homologs but ALKBH2 and ALKBH3 codes for functional nuclear DNA repair enzymes and protect cells from DNA damage following acute inflammation (Aas et al. 2003; Calvo et al. 2012). The AlkB enzyme belongs to non-heme Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases superfamily. These enzymes catalyze broad range of oxidation reactions (Fedeles et al. 2015). For example, Tet1–3 (Ten-eleven translocation) proteins reverse epigenetic modification 5mC to 5-hydroxymethyl-cytosine and further to 5-formyl-cytosine and 5-carboxyl-cytosine (Tahiliani et al. 2009; Ito et al. 2010; He et al. 2011; Ito et al. 2011; Pfaffeneder et al. 2011); AlkB homolog 5 (ALKBH5) and FTO (Jia et al. 2008; Lando et al. 2012; Zheng et al. 2013; Zhang et al. 2016) remove N6-methyladenosine (N6-meA). Besides nucleic acid modification, Fe(II)/2OG-dependent dioxygenases carry out variety of cellular functions, including post-translational modification of collagen proteins by prolyl hydroxylases (Ricard-Blum 2011); synthesis of quinolinic acid from 3-hydroxyanthranilic acid in the tryptophan-NAD+ biosynthetic pathway by metabolic enzyme 3-hydroxy-anthranilate dioxygenase (Brkic, Kovacevic and Tomic 2015); oxidative modification of the nuclear factor κB transcription factor by nuclear protein Pirin acts as (Liu et al. 2013), and so on. Structurally these proteins are characterized by eight antiparallel β-strands forming two β-sheets that commonly referred to as jelly-roll motif. Between the two β-sheets, an Fe(II) atom is bound via three conserved residues: a histidine, an aspartate/glutamate and a histidine (HXD/E…H). These enzymes require Fe2+, 2OG, O2 and ascorbate. AlkB family of Fe(II)/2OG-dependent dioxygenases are known to be regulated by availability of cofactors, including 2OG, oxygen and iron. For example, Tet enzymes, AlkB human homologs and histone demethylases are susceptible to inhibition by 2-hydroxyglutarate (2HG), a metabolite which is structurally similar to 2OG and accumulates in tumor cells due to mutations in isocitrate dehydrogenase (Xu et al. 2011; Wang et al. 2015). AlkB family of enzymes is also inhibited by lack of oxygen during cellular hypoxia (Anindya 2017). Although it has been speculated that availability of iron might influence the activity of Fe(II)/2OG-dependent dioxygenases family of enzymes (Mole 2010; Salminen, Kauppinen and Kaarniranta 2015), but to our knowledge no experimental or clinical studies exist to support this. In this paper, we examined how the cellular iron level impacts the function of Fe(II)/2OG-dependent dioxygenases family of enzymes involved in the DNA repair. We studied bacterial AlkB, S. cerevisiae AlkB homolog Tpa1 and human AlkB homolog AlkBH3, which are involved in alkylated DNA repair. Using previously described genetic systems we show that maintenance of iron homeostasis by transcription factor Aft1/Aft2 is crucial for the function under iron depletion condition. Our results suggest that function of Fe(II)/2OG-dependent dioxygenases family of enzymes will be affected if cellular iron level is not maintained in response to environmental conditions. MATERIALS AND METHODS Yeast strains and media Saccharomyces cerevisiae W3031a and its isogenic strains used in this study are described in Table 1. All knockout mutants were generated by using the one-step PCR method. Auxotrophic marker genes HIS3, TRP1, URA3 and LEU2 were amplified from pRS300 series vectors by using specific primers. Cells were grown in YPD or synthetic minimal media. To integrate the functional genes into yeast genome, counterselection method was used by selecting ura3 mutant cells on minimal media containing 5-fluoroorotic acid. Table 1. Saccharomyces cerevisiae strains used in this study. S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  View Large Table 1. Saccharomyces cerevisiae strains used in this study. S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  S. no.  Strain name  Genotype  1  W3031a  MATa (leu2–3112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15)  2  mag1Δ  mag1::TRP1  3  tpa1Δ  tpa1::HIS3  4  mag1Δtpa1Δ  mag1::TRP1tpa1::HIS3  5  aft1Δaft2Δ  aft1::TRP1 aft2::HIS3  6  mag1Δaft1Δaft2Δ  mag1::URA3 aft1::TRP1 aft2::HIS3  8  mag1ΔALKB  mag1::TRP1 URA3::ALKB  9  mag1ΔALKBH3  mag1::TRP1 URA3::ALKBH3  10  mag1Δaft1Δaft2ΔALKB  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKB  11  mag1Δaft1Δaft2ΔALKBH3  mag1::LEU2 aft1::TRP1 aft2::HIS3 URA3::ALKBH3  View Large Spot assay SD minimal medium was prepared by adding the appropriate amino acids to Yeast Nitrogen Base (YNB) without amino acids and supplemented with 2% glucose. This medium contains 0.2 mg/L iron from YNB; therefore, different concentrations of ferrozine (1–2 mM) were added for iron depletion. Approximately 6 × 106 cells were taken from exponentially growing culture, washed and suspended in 1 ml of potassium phosphate buffer pH 7.4. Seven microliters of 10-fold serially diluted four dilutions were spotted onto minimal media containing different concentrations of DNA damaging agents MMS or MNNG that supplemented without or with 1 mM ferrozine. Growth was monitored at 30°C for 5 days. Survival assay Cells were grown at 30˚C for overnight from a single colony in SD media containing 1 mM ferrozine. Thereafter, cells were treated with different concentrations of MMS. Approximately 4 × 103 cells were spread onto YPD medium and grown at 30˚C for 72 h. Cell survival was monitored by the number of colonies that were present. The number of colonies from untreated cells was taken as 100% and the percentage of cell viability was plotted by using Graph Pad-Prism software. RESULTS AND DISCUSSION Iron deficiency leads to inactivation of Tpa1 function Saccharomyces cerevisiae AFT1 gene is required for growth in iron-depleted media (Yamaguchi-Iwai, Dancis and Klausner 1995). As a first step in our analysis of growth under iron-limiting and iron-depleted condition, we examined growth of aft1Δ, aft2Δ and aft1Δ aft2Δ strains. We have used SD minimal medium, which contains low level of iron (0.2 mg/L). We have used the increasing concentration of iron chelator ferrozine (1–2 mM) to deplete iron and monitored growth of these strains by spot dilution assays on SD agar plates. As shown in Fig. 1A, aft1Δ, aft2Δ and aft1Δ aft2Δ strains showed cell growth comparable to wild-type when ferrozine concentration was between 1 and 1.5 mM, suggesting limited availability of iron in the media. In this study, we refer this experimental condition as iron-limiting. However, when ferrozine concentration was increased to 1.75 mM and above, aft1Δ aft2Δ strains showed growth defect, suggesting severe iron depletion under these conditions. Single-mutant strains aft1Δ, aft2Δ did not show any sensitivity to iron depletion under present experimental condition suggesting overlapping function of Aft1 and Aft2, as also observed previously (Blaiseau, Lesuisse and Camadro 2001). Figure 1. View largeDownload slide Deletion of genes encoding Aft1 and Aft2 lead to MMS sensitivity in an tpa1mag1Δ background. (A) Mild growth defect phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), aft1Δ, aft2Δ and aft11Δaft2Δ strains were spotted on SD supplemented with increasing concentrations of iron chelator ferrozine to deplete iron. The plates are shown after 4 days of incubation at 30°C. (B) Normal growth phenotype of aft11Δaft2Δ in the presence of MMS. Wild-type strain (W303), aft1Δ, aft2Δ, aft11Δaft2Δ and tpa1Δ strains were spotted on SD media containing DNA alkylating agent MMS as before. (C) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of ferrozine. Wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, mag1Δaft1Δ and mag1Δaft1Δaft2Δ strains were spotted on SD media containing either MMS (middle) or iron chelator ferrozine (left) or both (right) as before. (D) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of iron chelator curcumin. Wild-type and mutant strains were spotted on SD media containing MMS and iron chelator curcumin as before. (E) Normal growth phenotype of wild-type and mutant strains in the presence of ferrozine and SN1 alkylating agent MNNG. Strains were spotted on SD media containing either DNA alkylating agent MNNG (middle) or iron chelator ferrozine (left panel) or both (right panel). Figure 1. View largeDownload slide Deletion of genes encoding Aft1 and Aft2 lead to MMS sensitivity in an tpa1mag1Δ background. (A) Mild growth defect phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), aft1Δ, aft2Δ and aft11Δaft2Δ strains were spotted on SD supplemented with increasing concentrations of iron chelator ferrozine to deplete iron. The plates are shown after 4 days of incubation at 30°C. (B) Normal growth phenotype of aft11Δaft2Δ in the presence of MMS. Wild-type strain (W303), aft1Δ, aft2Δ, aft11Δaft2Δ and tpa1Δ strains were spotted on SD media containing DNA alkylating agent MMS as before. (C) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of ferrozine. Wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, mag1Δaft1Δ and mag1Δaft1Δaft2Δ strains were spotted on SD media containing either MMS (middle) or iron chelator ferrozine (left) or both (right) as before. (D) Hypersensitivity of aft11Δaft2Δtpa1Δ to MMS-induced DNA damage in the presence of iron chelator curcumin. Wild-type and mutant strains were spotted on SD media containing MMS and iron chelator curcumin as before. (E) Normal growth phenotype of wild-type and mutant strains in the presence of ferrozine and SN1 alkylating agent MNNG. Strains were spotted on SD media containing either DNA alkylating agent MNNG (middle) or iron chelator ferrozine (left panel) or both (right panel). Having established a condition where iron level is limiting but not totally depleted, we wanted to examine the role of iron-responsive transcription factor Aft1/Aft2 on the activity of Fe(II)/2OG-dependent dioxygenases. We hypothesized that under iron-limiting condition, Fe(II)/2OG-dependent dioxygenases involved in alkylated-DNA repair might exhibit reduced activity causing MMS sensitivity. In S. cerevisiae, MMS-induced DNA damage is repaired synergistically by the two different pathways, namely direct repair by Fe(II)/2OG-dependent dioxygenase Tpa1 and BER DNA glycosylase Mag1 (Shivange et al. 2014). It was also observed previously that the tpa1Δ deletion mutant had little MMS sensitivity at low concentration of MMS, due to Mag1 activity. Similarly, mag1Δ deletion also caused only modest MMS sensitivity. However, the double mutant tpa1Δ mag1Δ was hypersensitive to MMS (Shivange et al. 2014). We reasoned that under iron-limiting condition and in the absence of Aft1/Aft2, mag1Δ mutant cells would display MMS hypersensitivity akin to tpa1Δ mag1Δ cells, due to functionally defective Tpa1. Therefore, we generated aft1Δ, aft2Δ and aft1Δaft2Δ deletion strains and compared their survival with tpa1Δ strain at low concentration of MMS (0.0075%) by spot dilution assays on SD agar plates. As shown in Fig. 1B, none of these mutants were sensitive to low concentration of MMS used, suggesting that Aft1/Aft2 had no iron-independent and direct mechanism of causing MMS sensitivity. Having confirmed that aft1Δ, aft2Δ and aft1Δaft2Δ deletion strains being not sensitive to low dose of MMS, we made aft1Δmag1Δ and aft2Δmag1Δ double mutant and aft1Δaft2Δmag1Δ triple mutant strains and compared MMS sensitivity with tpa1Δmag1Δ strain under different conditions (Fig. 1C). As expected, wild-type cells grew in the presence of 0.0075% MMS, whereas mag1Δtpa1Δ mutants cells were extremely sensitive, in agreement with previous result (Shivange et al. 2014). Notably, aft1Δaft2Δmag1Δ showed stronger MMS sensitivity compared to aft1Δmag1Δ and aft2Δmag1Δ double mutant when cells were gown in the presence of 1 mM ferrozine. This growth defect is due to MMS exposure as without MMS, aft1Δaft2Δ mutant strain exhibited normal growth (Fig. 1C, left panel). Another iron chelator that is known to suppress the growth of S. cerevisiae is curcumin, a yellow pigment found in Indian herb turmeric (Minear et al. 2011). We performed growth assay with different concentrations of curcumin and found that at 50 μM concentration of curcumin, growth of wild-type and mutant yeast cells was not affected (Fig. 1D). The concentration of curcumin used here was significantly lower than what was reported to deplete cellular iron level (Azad et al. 2013) and thus could be considered iron-limiting. Next we investigated the effect of low concentration of MMS in the presence of curcumin. We observed that wild-type cells grew in the presence of 0.0075% MMS and 50 μM curcumin, whereas mag1Δtpa1Δ mutant cells were extremely sensitive, as expected. Interestingly, aft1Δaft2Δmag1Δ showed MMS sensitivity akin to mag1Δtpa1Δ mutants and mag1Δ single mutant was much less sensitive. These data support our hypothesis that when iron is limiting, iron-responsive transcription factors are needed to maintain cellular iron level and thereby keeping the Fe(II)/2OG-dependent dioxygenase Tpa1 active. Lack of iron homeostasis, however, rendered Tpa1 inactive under the condition where iron was limiting. It was reported earlier that AlkB family members repair only the N-alkylation damage caused by SN-2 type of alkylating agent MMS, but not involved in repair of the O-alkylation adducts such as O6-meG caused by SN-1 type of alkylating agent 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG). In order to examine if iron depletion leads to sensitivity to O-alkylating agents, we analyzed cell survival in the presence of MNNG by spot dilution method on SD agar plates. As shown in Fig. 1D, both aft1Δaft2Δmag1Δ and tpa1Δmag1Δ strains could tolerate 15% of MNNG due to active DNA repair. As Tpa1 and Mag1 are not involved in repairing MNNG-induced damage, double mutant tpa1Δamag1Δ strain did not show any sensitivity to MNNG compared to wild type (Fig. 1E). This result confirms that MMS hypersensitivity of aft1Δaft2Δmag1Δ cells could be due to lack of Tpa1-mediated repair under iron-limiting condition. Iron deficiency leads to inactivation of bacterial AlkB and human AlkBH3 function Saccharomyces cerevisiae Fe(II)/2OG-dependent dioxygenase Tpa1 is a functional ortholog of bacterial AlkB. Although there are nine AlkB family proteins in human, AlkBH2 and AlkBH3 are bona fide nuclear DNA repair enzymes. We hypothesized that when iron depletion could inhibit Tpa1 DNA repair activity, it would also similarly affect bacterial AlkB or human AlkB orthologs. Therefore, we decided to study two more Fe(II)/2OG-dependent dioxygenases, namely E. coli AlkB and human AlkBH3 in S. cerevisiae under iron-limiting condition. We used a plasmid shuffle strategy to introduce the bacterial or human gene variant in the tpa1Δ mutants. Once we obtained the complementation strains, we wanted to test whether AlkB or AlkBH3 could rescue MMS hypersensitivity of mag1Δtpa1Δ strain and rescue the effect of deletion of Tpa1 on MMS sensitivity. For this, we performed spot dilution assays on SD agar plates containing a range of MMS concentrations from 0.0075% to 0.015%. Figure 2A shows that the tpa1Δmag1Δ strain is exquisitely sensitive to MMS, but when ALKB and ALKBH3 complemented TPA1, the strains were able to rescue the sensitivity to MMS. This restoration of resistance to MMS by AlkB and AlkBH3 suggests functional complementation. Next, we examined the effect of limiting iron concentration on the activity of AlkB and AlkBH3. For this, we introduced E. coli AlkB and human AlkBH3 in aft1Δaft2Δmag1Δtpa1Δ background. As shown in Fig. 2B, AlkB and AlkBH3 expressing cells showed strong MMS sensitivity of when cells were gown in the presence of 1 mM ferrozine. This growth defect is further pronounced in aft1Δaft2Δ genetic background. This increase in sensitivity to MMS is similar to that observed for aft1Δaft2Δmag1Δ strain. This result supports our hypothesis that lack of Aft1/Aft2 in the iron-limiting condition severely affects the activity of AlkB family of Fe(II)/2OG-dependent dioxygenases and results in MMS sensitivity. Figure 2. View largeDownload slide Functional complementation of S. cerevisiae Tpa1 by E. coli AlkB and human AlkBH3. (A) MMS sensitivity phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were spotted on SD media containing MMS. The plates are shown after 4 days of incubation at 30°C. (B) Hypersensitivity to alkylating agent MMS in the absence of genes coding Aft1 and Aft2. Wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were grown as before in the presence of MMS and iron chelator ferrozine. Figure 2. View largeDownload slide Functional complementation of S. cerevisiae Tpa1 by E. coli AlkB and human AlkBH3. (A) MMS sensitivity phenotypes of aft11Δaft2Δ mutant under iron depletion condition. Ten-fold dilutions of log-phase cultures of wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were spotted on SD media containing MMS. The plates are shown after 4 days of incubation at 30°C. (B) Hypersensitivity to alkylating agent MMS in the absence of genes coding Aft1 and Aft2. Wild-type strain (W303), mag1Δ, mag1Δ tpa1Δ, and AlkB and AlkBH3 expressing strains with mag1Δ tpa1Δ background were grown as before in the presence of MMS and iron chelator ferrozine. Iron deficiency results in reduced cell survival after exposure to MMS We quantified the ability of wild-type and mutant strains to form colonies following acute exposure to MMS. We treated the cells in SD media containing 0.005%, 0.015% and 0.030% MMS and 1 mM ferrozine and plated a fixed number of cells of each strain on YPD agar plates without MMS. The surviving colonies were counted and expressed as percent survival by comparing with untreated cells (Fig. 3A and B). There was no survival of aft1Δaft2Δmag1Δ and mag1Δtpa1Δ strains observed in SD medium supplemented with ferrozine and MMS. This result is in agreement with our earlier observation and suggests that Tpa1 function is affected under chronic iron-limiting condition and affects cell survival to MMS exposure. We have also quantified the ability of AlkB and AlkBH3 expressing strains to survive and colonies after acute exposure to MMS. We treated each of the strains in SD media containing 0.005%, 0.015% and 0.030% MMS and 1 mM ferrozine and plated a fixed number of cells on YPD agar plates without MMS. The surviving colonies were counted in each plate and expressed as percent survival by comparing to untreated cells (Fig. 3C). Strains expressing AlkB and AlkBH3 in aft1Δaft2Δ background were failed to grow similar to mag1Δtpa1Δ strains in SD medium supplemented with ferrozine and MMS (Fig. 3D). This result is comparable to Tpa1 expressing cells and suggests that function of AlkB and AlkBH3 is equally affected under chronic iron-limiting condition in the absence of iron homeostasis by Aft1/Aft2. Figure 3. View largeDownload slide Survival analysis of strains expressing S. cerevisiae Tpa1 or E.coli AlkB or human AlkBH3. (A) Survival curve of wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, aft1Δaft2Δ and aft11Δaft2Δmag1Δ strains grown in cultures containing 0.05%, 0.15% and 0.30% (v/v) of methylating agent MMS. Survival was determined by plating cells on LB agar at 37°C for 20 h and colony counting. The viability of cells not treated with MMS was 100%. Error bars indicate the standard error of the mean. All the strains are the isogenic derivatives of W303. (B) Assessment of MMS sensitivity of tpa1Δmag1Δ and aft11Δaft2Δmag1Δ strains. Colonies of each strain were streaked on an SD plate containing iron chelator ferrozine and either no MMS or 0.0075% (v/v) MMS. As a control, wild-type strain (W303) was streaked. Plates were incubated at 30°C for 4 days and photographed. (C) Survival curve of strains expressing E. coli AlkB and human AlkBH3. Cell viability was determined as before. (D) Assessment of MMS sensitivity of E. coli AlkB or human AlkBH3 in aft1Δaft2Δ background. All treatments were as described in B. Error bars represent the SEM of six replicates from three representative experiments that were repeated two times. *P < 0.05, ANOVA. Figure 3. View largeDownload slide Survival analysis of strains expressing S. cerevisiae Tpa1 or E.coli AlkB or human AlkBH3. (A) Survival curve of wild-type strain (W303), mag1Δ, mag1Δtpa1Δ, aft1Δaft2Δ and aft11Δaft2Δmag1Δ strains grown in cultures containing 0.05%, 0.15% and 0.30% (v/v) of methylating agent MMS. Survival was determined by plating cells on LB agar at 37°C for 20 h and colony counting. The viability of cells not treated with MMS was 100%. Error bars indicate the standard error of the mean. All the strains are the isogenic derivatives of W303. (B) Assessment of MMS sensitivity of tpa1Δmag1Δ and aft11Δaft2Δmag1Δ strains. Colonies of each strain were streaked on an SD plate containing iron chelator ferrozine and either no MMS or 0.0075% (v/v) MMS. As a control, wild-type strain (W303) was streaked. Plates were incubated at 30°C for 4 days and photographed. (C) Survival curve of strains expressing E. coli AlkB and human AlkBH3. Cell viability was determined as before. (D) Assessment of MMS sensitivity of E. coli AlkB or human AlkBH3 in aft1Δaft2Δ background. All treatments were as described in B. Error bars represent the SEM of six replicates from three representative experiments that were repeated two times. *P < 0.05, ANOVA. It was also published before that when S. cerevisiae cells are grown under iron-depleted condition, mRNA levels of several proteins containing Fe-S cluster proteins, such as aconitase and succinate dehydrogenase, are downregulated. Although Fe(II)/2OG-dependent dioxygenases like Tpa1 are iron-containing enzymes, they bind non-heme Fe(II) and not the Fe-S cluster. Furthermore, multiple microarray studies carried out to determine the genes regulated by iron levels revealed that mRNA level of Tpa1 is not downregulated under low iron conditions (Shakoury-Elizeh et al. 2004; Berthelet et al. 2010). TPA1 promoter does not have recognition sequence for Aft1/Aft2 nor were they reported to be part of ‘iron regulon’ genes (Philpott et al. 2002; Philpott and Protchenko 2008). Therefore, our data presented here indicate that in the absence of Aft1/Aft2, lack of iron homeostasis is most likely responsible for MMS hypersensitivity of aft1Δaft2Δmag1Δ strain. In the absence of iron responsive transcription factor Aft1, S. cerevisiae is sensitive to high dose of MMS (0.05%) when grown in completely iron-depleted media (Berthelet et al. 2010). This MMS sensitivity was restored by exogenously supplying iron. It remains completely unknown how Aft1 might influence MMS sensitivity. Taken together, our data suggest that depletion of iron renders Fe(II)/2OG-dependent dioxygenases inactive and support the hypothesis that cellular iron concentration could be an important regulator of AlkB family of enzymes. Limited bioavailability has been shown to affect Fe(II)/2OG-dependent dioxygenases family of enzyme prolyl-hydroxylase involved in the degradation of hypoxia inducible factor (Knowles et al. 2006; Pan et al. 2007). Indeed, individuals with severe iron-deficient anemia were reported to have impaired hypoxic response (Frise et al. 2016). Our results suggest that function of Fe(II)/2OG-dependent dioxygenases family of DNA repair enzymes will be affected if cellular iron level is not maintained in response to environmental conditions. Because iron deficiency affects vast number individuals worldwide, DNA repair defect due to iron deficiency could be of considerable importance for human health and disease. 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FEMS Yeast ResearchOxford University Press

Published: Mar 1, 2018

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