Proteome analysis of Saccharomyces cerevisiae after methyl methane sulfonate (MMS) treatment

Abstract

The treatment of methyl methane sulfonate (MMS) increases sensitivity to the DNA damage which, further leads to the cell death followed by a cell cycle delay. Delay in the cell cycle is because of the change in global transcription regulation which results into proteome change. There are several microarray studies on the transcriptome changes after MMS treatment, but very few studies are reported related to proteome change. The proteome analysis in this report identified subgroups of proteins, belonging to known cell cycle regulators, metabolic pathways and protein folding. About 53 proteins were identified by MS/MS and found that 36 of them were induced, 10 were repressed and few of them showed insignificant change. Our results indicated the change in the interactome as well as phosphorylation status of carboxy terminal domain (CTD) of RNA Polymerase II (RNAP-II) after MMS treatment. The RNAP-II complex was affinity purified and ~1640 peptides were identified using nano LC/MS corresponding to 27 interacting proteins along with the twelve RNAP-II subunit. These identified proteins participated in the repair of the damage, changes the function of the main energetic pathways and the carbon flux in various end products. The main metabolic enzymes in the glycolysis, pyruvate phosphate and amino acid biosynthesis pathways showed significant change. Our results indicate that DNA damage is somehow related to these pathways and is co-regulated simultaneously.

Highlights

• •The MMS treatment leads to some major metabolic pathways changes.
• •The MMS treatment showed the hyperphosphorylation of RNAP-II CTD.
• •The changes in the phosphorylation leads to change in the interactome of RNAP-II.

1. Introduction

Methyl methane sulfonate (MMS) is an alkylating agent as well as a potential carcinogen [1]. It methylates N3 and N7 position of deoxy-adenosine and deoxy-guanosine respectively as well as other oxygen and nitrogen atoms of the nitrogenous bases [2]. The modifications by MMS lead to double stranded DNA breaks or DNA damage. The presence of damage is signaled through specific phosphorylation pathways which further activate DNA damage response genes. Some of these pathways involve the yeast protein kinases Mec1 and Dun1 that eventually alter the activity of transcription factors [3,4]. These transcription factors regulate the whole genome expression. Due to changes in the activity of transcription factors set of genes are induced and repressed that rebuild and repair the damage [5,6]. The DNA damage produced by the MMS leads to cause cancer. So it is important to identify the global set of genes induced and repressed in response to damage for defining the cellular pathway involved in the recognition and repair of damage. The study of DNA damage on cells can give us the understanding of the controlling factor which governs the whole process and the pool of the genes affected [7].

DNA damage and their governing factors have been studied in a number of ways. In Saccharomyces cerevisiae the transcriptional response at multiple time point has been assayed in presence of MMS using microarray [8,9]. Gasch et al studied transcriptional response in S. cerevisiae and reported more than 750 genes. His group identified cluster of genes co-regulated temporally in response to MMS [10,11]. In similar experiments Jelinsky et al reported 693 genes showed a significant transcriptional modulation in response to MMS [9]. In every new study additional genes have been identified as having a role in damage response. There is no single technique which can investigate the whole process so different approaches have been used.

There are some proteins which regulate the transcription by the phosphorylation of the carboxy terminal domain (CTD) of RNA Polymerase-II (RNAP-II). The CTD acts as a platform for the recruitment of transcription regulatory proteins [[12], [13], [14], [15]]. The phosphorylation of the CTD exists in multiple forms [16]. Bensaude and colleagues first observed the accumulation of the phosphorylated IIo form of the CTD in case of the heat shock. Further experiments in Saccharomyces cerevisiae show the rapid increase in Ser2 phosphorylation (Ser2P), but no change in Ser5 phosphorylation (Ser5P) in the heptad of RNAP II CTD [17]. Global changes in CTD phosphorylation are also brought about by DNA damaging agents [18]. The change in the CTD phosphorylation level bought together in several ways first may be the activation of the several kinases like Bur1 and Ctk1 in Saccharomyces cerevisiae which leads to Ser2P [19]. Increase in the Ser2P leads to the depletion of the RNAPII available for initiation so stress response leads to the reduction in gene expression. Finally, gene expression is controlled globally and at multiple levels in response to MMS stress. The relationships among these global regulations are still unclear.

Here in this study, we analyzed global regulation during MMS stress using proteomics approaches. We obtained a global overview of changes in proteome under MMS stress using two-dimensional gel electrophoresis (2-DE). We also checked the change in the CTD phosphorylation and the interactome of RNAP-II in MMS stress condition. Furthermore, the interactome of RNAP-II was identified by nano LC/MS using in solution trypsin digestion of the purified complex in normal as well as in MMS treated condition. The data indicate that in stress condition, there are changes in the proteome as well as in the proteins which are directly or indirectly interacting with the RNAP-II complex. The potential utility of this approach results in the detection of both known and novel DNA damage responsive proteins.

2. Materials and methods

2.1. Spot assay and growth curve

Spotting assay was done as described somewhere [20]. The Saccharomyces cerevisiae BY4741 strain was grown overnight in YPD at 30 °C on 200 rpm shaking. Secondary culture was done and cells were grown till OD₆₀₀ 0.7. For each condition equal numbers of cells were pellet and serial dilution of 10 fold was made till 10⁻⁵ dilution. Then 10 μL aliquots from each of the dilutions were spotted on plates containing different concentrations of MMS (0.005, 0.001, 0.02, or 0.05%). The plates were incubated for 2–3 days at 30 °C and 25 °C. For growth curve, cells were grown in similar condition as mentioned and at every hour OD₆₀₀ was calculated and plot against incubation time.

2.2. Sample collection and sample preparation

The Saccharomyces cerevisiae BY4741 strain was grown in YPD medium till 0.7 OD₆₀₀ then 0.02% MMS treatment was done for 6 h. The cells were collected by centrifugation at 5000 rpm for 5 min and washed with phosphate buffered saline (PBS, pH7.4). Approximately 500 mg cells were suspended in 2.0 ml of protein extraction buffer (20 mM Tris, 4% CHAPS, 2.5% glycerol and 1X PIC). Cells were lysed using French press (30 Kpsi) and the supernatant was collected by centrifugation at 14,000 rpm for 30 min at 4 °C. The proteins in the supernatant were precipitated with 10% (v/V) TCA and incubated overnight at −20 °C. The precipitated protein was collected after centrifugation at 14,000 rpm for 15 min. The pellets were washed thrice with ice chilled acetone. Finally, the protein pellet was suspended in 300 μl of rehydration buffer (7 M urea, 2 M thio-urea, CHAPS 4%, DTT 10 mM, Tris 20 mM). Pellets were briefly sonicated in rehydration buffer and incubated for 2 h. Protein concentration was estimated and samples were stored at −80 °C. Approximately, 250 μl rehydration solution containing 250 μg protein was applied in rehydration tray and IPG strip (nonlinear, 11 cm, 3–10 pH range) was placed on rehydration solution mixture for strip rehydration. Samples were rehydrated passively for overnight in rehydration tray (GE healthcare).

2.3. IEF run and 2D electrophoresis

Rehydrated IPG strips were run on Ettan IPG phor3 (GE healthcare). After completion of IEF, the strips were equilibrated in equilibrium buffer (6 M Urea, 75 mM Tris-Cl (pH 8.8), 29.3% Glycerol, 2% SDS, 1% BPB). First equilibrium was performed with 100 mg DTT supplemented with 10 ml Equilibrium buffer followed by second equilibrium with the addition of 250 mg iodoacetamide in 10 ml Equilibrium buffer. Each equilibrium step was performed for 30 min. Equilibrated strips were placed at 12%, 18 × 16 cm acrylamide gel for second dimension gel electrophoresis. The gel was overlaid with 0.5% agarose prepared in 1X TGS buffer and was run at constant current of 40 mA for 6–7 h till dye reached to bottom. The gel was stained using coomassiae brilliant blue R-250 (CBB R-250) solution (0.25% CBB R-250, 45% methanol, 10% glacial acetic acid) for overnight at room temperature and then destained in destaining solution (45% methanol, 10% glacial acetic acid). The gel was scanned with Labscan software (GE healthcare) and analysis was performed by Image Master^TM 2D Platinum 7.0.

2.4. Identification of differentially expressed protein by MS/MS analysis

Differentially expressed protein spots from 2D gel were excised, chopped and destained. The gel pieces were dehydrated with solution A (2:1 mixture of acetonitrile (ACN): 50 mM ABC) and then rehydrated with 50 mM Ammonium Bicarbonate (ABC). Proteins in the gel pieces were reduced with 10 mM DTT (56 °C, 30 min in the dark), alkylated with 50 mM iodoacetamide (room temperature, 30 min) and digested with trypsin in 25 mM ammonium bicarbonate buffer (pH 8.0) at 37 °C for overnight. The digested peptides were extracted in extraction solution 50% ACN and 0.1% TFA in total volume 100 μl and speed vacuum to make the volume up to 5–10 μl. 0.5 μl of extracted peptides were spotted on the MALDI plate followed by 0.5 μl α-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 50% ACN and 0.5% TFA). Spots were dried completely and used for MS-MS on MALDI-TOF-TOF platform (model 4800, ABsciex, USA). The result obtained from MS/MS were analyzed by MASCOT search using NCBInr databases with following parameter (fixed precursor ion mass tolerance of 20 ppm, fragment ion mass tolerance of 0.05 Da, calibration error of 0.005 Da, one missed cleavage, carbamido methylation of cysteines and possible oxidation of methinonine).

2.5. Protein identification using nanoLC/MS

TAP purification was done as described in Tardiff et al [21]. The in-solution trypsin digestion of the purified protein complex was done. Chromatographic separations of peptides were done on a nano-LC system consisting of a Nano-LC trap (3 μm, ChromXP C18CL, 120 Å, 0.5 mm × 350 μm (AB Sciex)) and a reversed-phase analytical (Chromolith Cap Rod RP-18e (150 × 0.1 mm) monolithic capillary column [22]. Tryptic digested samples were injected by auto sampler into the column. Washing of the trap column with solvent “1A” (2% ACN, 98% dH2O and 0.1% TFA) for 5 min at a flow rate of 10 μL/min was done for the de-saltification of the peptide mixture. Desalted peptide separation was achieved using solvent “2A” (2% ACN, 98% dH2O and 0.1% TFA) and “2B” (98% ACN, 2% dH2O and 0.1% TFA) with a flow rate of 800 nL/min in a linear gradient elution mode ramping from 0% to 90% “2B” over the course of 110 min. Peptide fractions were mixed online with α-cyano-4-hydroxycinnamic acid matrix solution (5 mg/mL in 50% ACN + 0.1% TFA) in a ratio 1:1 (v/v) and spotted onto a 384 wells containing stainless steel MALDI target plate (AB Sciex) [23]. Automatic spotting time was set from 5 min to 110 min, with 20 s drop intervals.

2.6. Mass spectral and data analysis

MS and MS/MS analysis were carried out on a 4800 MALDI-TOF/TOF mass spectrometer (AB SCIEX, USA) equipped with 355 nm Nd:YAG solid state laser. Positive ion mass spectra were recorded in reflectron mode over a mass range of m/z 800–4000 Da. External mass calibration was performed using TOF/TOF calibration mixture (des-Arg1-bradykinin, [M+H]+ = 904.468 Da; Angiotensin I, [M+H]+ = 1296.685 Da; Glu1-fibrinopeptide B, [M+H]+ = 1570.677 Da; ACTH (1–17 clip), [M+H]+ = 2093.087 Da; ACTH (18–39 clip), [M+H]+ = 2465.199 Da, and ACTH (7–38 clip), [M+H]+ = 3657.9294 Da). A maximum of twenty five precursors from each mass spectrum with a minimum signal/noise ratio of fifteen were selected for MS/MS analysis. Analysis of MS and MS/MS data and database searching were conducted against the NCBInr database using ProteinPilot software (Version. 4.0, AB Sciex) with the MASCOT search engine using following search parameters (taxonomy was set to S. cerevisiae, trypsin as enzyme (one missed cleavage allowed), variable modification of Oxidation (M), precursor tolerance was set to 100 ppm and MS/MS fragment tolerance to 0.2 Da, and peptide charge was “1+“. Resulted proteins were considered at a significance level of p < 0.05).

2.7. CTD phosphorylation status and western blotting

To check the phosphorylation status of the CTD of RNAP-II the yeast strain was grown overnight in YPD broth. Then 500 ml YPD broth was inoculated with 1% of the overnight culture and grown at 30 °C and 200 rpm. After ~0.2–0.3 OD₆₀₀ culture was treated with 0.02% MMS. 50 ml of sample was collected at 0, 15, 30, 45, and 60 min time interval. After collecting the sample immediately centrifuge at 5000 rpm for 5 min and then pellet was washed with chilled millQ and store at −20 °C. Now the pellets were collected in 1.5 ml microfuge tube and washed with 1X PBS and then suspended in 100 μl 1X-PBS with 1X-PIC and 2 mM PMSF. Then samples were lysed using bead beater (20 s on bead beater and 3 min on ice, 3 shots). Now the lysed samples were collected in another 1.5 ml microfuge tube by pricking at the bottom of previous tube. Cell debris was removed by centrifugation (5000 rpm, 5 min) and the lysate were collected in another microfuge tube. Samples were quantified using a standard Bradford method using 10 mg/ml BSA as standard. 100 μg of protein was loaded on 8% SDS-PAGE. Then protein was transferred to nitrocellulose membrane and checked the rpb1 using anti-TAP (1:1000) and ser2, ser5, and ser7 phosphorylation was checked using 3E10 (1:1000), 3E8 (1:1000), 4E12 (1:1000). Secondary antibody anti mouse IgG HRP conjugated (1:5000) for anti TAP and anti-rabbit IgG HRP conjugated (1:5000) for phosphorylation [14,24].

3. Results

3.1. Yeast sensitivity to MMS treatment

The sensitivity of the strain at different MMS concentration (0.005, 0.01, 0.02, 0.05%) was checked. The results showed that increasing concentration of MMS lead to decrease cell viability. Finally, the higher concentration of MMS became lethal to cell. The growth of the cells in 0.02% MMS was very slow and ≥0.05% MMS concentration was found to be lethal even at 25 °C (Fig. 1A). Furthermore, we checked the growth of the strain in 0.02% MMS concentration in broth and found that the growth was slow as compared to untreated sample. A shift in the growth curve at the initial phase was also observed. After initial slow lag phase as compared to the control, the growth was recovered at later stage (Fig. 1B). The results indicated that 0.02% MMS concentration was optimal for the proteomics analysis.

Fig. 1.

MMS sensitivity of yeast cells at different temperature. (A) The cultures were grown in YPD until the mid-log phase and a serial tenfold dilution were prepared and spotted onto YPAD plates containing different concentrations of MMS and incubated for two days. (B) Growth curve of yeast cell in the presence and absence of 0.02% MMS at 30 °C.

3.2. Proteome analysis after MMS treatment

To understand the functional proteomics of the yeast in DNA-damage response, the whole cell proteome analysis was performed by 2-D gel electrophoresis (2DE) and identification of the differentially expressed proteins was carried out by MS/MS followed by in gel trypsin digestion. The yeast cell culture was grown till OD₆₀₀ of 0.7 and then treated with 0.02% MMS for 6 h, extraction of protein was done as described in the material and methods. The 2-DE was carried out in replicates to allow valid statistical analysis (Fig. 2). Upon scanning and analyses of the spots (each spot was given a spot ID in the image analysis) the proteins were identified by MS/MS. We choose 97 spots for the analysis; however, only 53 of them could be identified by MS/MS. Among the identified spots, 36 spots showed induced expression or up-regulation of the gene, 10 showed down regulation or repression, and 7 spots showed no change or insignificant change in the protein expression (Supplementary Table 1). A few proteins such as Eno2, Adh1, and Pdc1 etc were identified at multiple spots. These proteins undergo post translational modification [[25], [26], [27]] and hence exists in multiple forms with different molecular masses and the isoelectric point, and hence been identified at more than one position (Fig. 2).

Fig. 2.

Image of 2D gel (10% SDS-PAGE) labeled with the Spot ID and the name of the protein identified by MALDI. Red and black arrows represent the induced and repressed spots respectively, while orange arrow shows spot with no significant change in the expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. Functional characterization of identified proteins

The identified proteins were functionally divided into four different categories according to biological function (Fig. 3A). Most of the proteins belong to different metabolic pathways like glycolysis (14 proteins), pyruvate (4 proteins), phosphate pentose pathway/PPP (3 proteins) and amino acid metabolism pathway (4 proteins). Proteins related to DNA damage (6 proteins) and protein folding pathways (8 proteins) were also identified. Average fold change in expression of the proteins related to different pathways was calculated. The results indicated 3 fold changes in cell cycle regulators and PPP proteins while more than two fold change in the expression of the proteins related to protein folding and glycolysis (Fig. 3B).

Fig. 3.

Functional characterization of identifies proteins. (A) Diagrammatic representation of the identified proteins in 4 major metabolic pathways. (B) Average fold change in expression of the proteins related to a particular pathway (calculated by the average of fold change expression in total identified proteins in the specific pathway).

3.4. MMS treatment leads to the increase in the CTD phosphorylation

The expression of the protein is directly linked to the transcription of their respective genes which is regulated by the RNAPII and epigenetic modification (phospho/dephosphorylaltion) on the CTD [[12], [13], [14], [15]]. In order to further investigate the effect of MMS treatment on the phosphorylation status of RNAPII-CTD the western blotting was done using phosphospecific antibodies against CTD [28,29]. The results showed that after 30 min MMS treatment the phosphorylation of Ser2 was higher as compared to the untreated sample (Fig. 4A, right side). The Ser5 phosphorylation level was almost same in all the samples but hyper-phosphorylation (IIo) level was higher in treated samples as compared to hypo-phosphorylation level (IIa) while in control samples the IIa level was higher as compared to IIo level. The same results was found in case of Ser7 phosphorylation where IIo form was higher in treated condition, but IIa form was higher in the control condition. The time dependent increase in the phosphorylation level was also observed (Fig. 4A, right side). For reference, Rpb1 level was checked in each sample (Fig. 4A, left side). This indicates that cells respond to DNA damage by inducing the CTD phosphorylation as well as by increasing hyper-phosphorylation level [30,31]. The findings in this regard suggest that alterations in CTD phosphorylation may play an important role in the activities of RNAP-II, adjusting it to stressed conditions [30,32].

Fig. 4.

CTD phosphorylation leads to change in the interactome of RNAP-II. (A) Cells were grown till 0.8 OD and treated with 0.02% MMS and the samples were collected at different time interval. Western blotting was done to check the Ser2, Ser5 and Ser7 phosphorylation status using 3E10, 3E8 and 4E12 antibody, respectively. Rpb1 level was checked using anti-TAP antibody. (B) Schematic representation of the strategies for the identification of the RNAP-II associated proteins. (C) 10% SDS-PAGE where Lane1 represent the ladder and lane2, 3 and 4 represent RNAP-II purification from JTY1, Y1056 and Y1088 respectively.

3.5. The MMS treatment leads to changes RNAP-II interactome

As it is evident from the earlier experiment, that MMS treatment altered the phosphorylation status of the CTD. Furthermore, the question remains whether the change in the CTD phosphorylation upon MMS treatment leads to change in the interactome of RNAP-II or not. To investigate this, the TAP purification of RNAPII was performed as described in Tardiff et al [21]. The strategy of the RNAPII complex purification and their identification is schematically represented in Fig. 4B. The purified complex was identified using two different approaches. In the first approach in-gel trypsin digestion was done, the peptides were purified from the gel and then MS/MS identification was done. On the other hand, in second approach in-solution trypsin digestion of purified complex was done and directly loaded on the nanoLC column and identified by MS/MS. TAP purification was done using Y1056 strain (Fig. 4C, lane 3). JTY1 strain having no TAP tag was taken as a control (Fig. 4C, lane 2). After purification, the complex was run on SDS-PAGE (Fig. 4C) and the bands were identified by MS/MS after in-solution trypsin digestion (Fig. 4B). Using first approach Spt5, Tsa1 and Ssa1 proteins along with the 12 subunits of RNAPII were identified. For confirmation, the same complex was identified using a different strain Y1088 which was having TAP as well as HBH tag (Fig. 4C, lane 4). On the other hand using second approach the RNAP-II complex was identified from the same sample. The ~660 peptides were obtained by nano LC/MS after in-solution trypsin digestion which represented 12 subunits along with few interacting proteins. In order to find out the change in interactome after MMS stress the culture with treated with 0.02% MMS at 0.7 OD₆₀₀ for 6 h and the complex was identified as described earlier. Approximately 1640 peptides were identified which correspond to RNAP-II subunits and 27 interacting proteins which are listed in the Table 1. The group of proteins included all core RNAP-II subunits as expected already identified by Tardiff et al [21]. More numbers of proteins were identified as compared to the earlier report (Table 1).

Table 1.

Proteins identified after TAP purification of Y1056 strains using nano LC/MS data and its comparison with that of identified by Tardiff et. al., 2007.

Control		Treated (MMS)			Tardiff et al., 2007
Rpb1 Rpb2 Rpb3 Rpb4 Rpb5 Rpb6 Rpb7 Rpb8 Rpb9 Rpb10 Rpb11 Rpb12 Spt5	Hsp70 Tsa1 Ssa1	Rpb1 Rpb2 Rpb3 Rpb4 Rpb5 Rpb6 Rpb7 Rpb8 Rpb9 Rpb10 Rpb11 Rpb12 Spt5	Tcp1 Ssb2 Nop58 Tdh3 Tdh2 Adh1 Cad1 Rpp0 Rtr1 Rps2 Kar2 Sam1 Tip49 Spt4	Spt6 Lsm1 Iwr1 Ssf2 Kch1 Npa1 Ire1 Adh2 Cct7 Ssa2 Ssb1 Tef2 TFIIF Tfg2	Rpb1 Rpb2 Rpb3 Rpb4 Rpb5 Rpb6 Rpb7 Rpb8 Rpb9 Rpb10 Rpb11 Rpb12 Spt5	Tfg1 Tfg2

The identified proteins were analyzed by network mapping using the String 10 server [33]. The network map showed that most of the identified proteins were associated with the basal transcription factors and RNA biogenesis (Fig. 5). The protein belonging to the basal transcription factors includes Tgf1, Tgf2, Spt5, Irw1 and Rtr1 while Rvb1, Rvb2, Nop58, Nop1, Cbf5 belongs to the RNA biogenesis. Some other factors (Ire1, Cad1, Lsm1 and Lsm7) were also identified irrespective of these categories. Basal transcription factors as well as RNA biogenesis factors are responsible for the global transcription regulation. Spt5 which is the component of DSIF complex was present in stress as well as control condition which functions in various aspects of transcription, chromatin maintenance, and RNA processing [34,35]. The attachment of the TFIIF with the RNAPII assembly was observed in case of treatment which was not present in the control sample.

Fig. 5.

Network mapping of the RNAPII associated protein using string 10. The nodes represent the protein. The empty node represents the protein without 3D structure while the filled node represents the protein with known or predicted 3D structure. The colorful lines are called edges which represents the protein-protein interaction. The edges show the number of evidence of protein-protein interactions. The network mapping shows the proteins related to RNA biogenesis and basal transcription factors.

4. Discussion

4.1. MMS treatment leads to activation of the DNA damage response pathway

The results proposed that MMS treatment mainly influences four metabolic pathways. The proteins related to pentose phosphate pathway (PPP), cell cycle and growth regulators were most affected (Fig. 3A and B). The identified proteins belong to checkpoint-regulated proteins, including Rnr4 and Bmh1 which play integral role in DNA replication and checkpoint control. Rnr4 is the small subunit of the ribonucleotide diphosphate reductase, which catalyzes the rate-limiting step in dNTP synthesis and is regulated by DNA replication and DNA damage checkpoint pathways via localization of the small subunits from nucleus to cytoplasm [36,37]. Rnr4 also controls the nucleotide pool for DNA synthesis and is a downstream target of the Rad53 checkpoint kinase [38]. On the other hand, Bmh1 controls proteome at a post-transcriptional level and involved in the regulation of Ras/MAPK and rapamycin-sensitive signaling [39,40]. Most of these proteins had been identified by Kim et al in the proteome analysis after chromatin enrichment (Table 2) [41]. Along with the previously well characterize proteins, several additional DNA damage response (DDR) associated proteins were identified including Vma1, Vma2, Vma4, Stm1, Cys3, and Tif1 which were differentially expressed in MMS stress. Proteome analysis showed that the level of Rnr4, Vma2, Vma1, Stm1 and Pre9 was found to be increased while the Vma4, Bmh1, Cys3 and Tif1 shows the little change in the expression of the protein (Supplementary Table 1).

Table 2.

Comparison of the identified proteins with the earlier studies. The proteins shown in different proteomics analysis have been categories as PA (the proteins which align with 2DE analysis data) and RNAPII (the proteins which align with RNAP-II interactome data).

This study		Chromatin enrichment Kim et al., 2011		Gasch et al., 2001		Jelinsky et al., 1999
(PA)	(RNAPII)	(PA)	(RNAPII)	(PA)	(RNAPII)	(PA)	(RNAPII)
Ipp1, Tdh2, Tdh3, Fba1, YNL134C, Adh1, Tif1, Tdh2, Eno2, Gnd1, Eno1, Vma2, Cys4, Pdc1, Vma1, Ssc1, Tkl1, Tpi1, Adk1, Vma4, RPS0A, Spe3, Fba1, Pgk1, Eno2, Gdh1, Pgi1, Frs1, Bmh1, Rpp0, Stm1, Rnr4, Ilv5, Ura1, Cys3, Hsp60, Pdc1, Ssb2, Trr1, Tsa1, Pre9, Hxk2, Hsc82, Gpm1, Gpd1, Sti1, Sse1, Oye3, Ssa1	Tcp1, Ssb2, Nop58, Tdh3, Tdh2, Adh1, Rpp0, Rtr1, Rps2, Sam1, Spt4, Spt6, Lsm1, Iwr1, Ssf2, Kch1, Npa1, Ire1, Adh2, Ssa2, Tef2, TFIIF, Tfg2, Tip49 (Rvb1), Ssb1, Kar2, Cad1, Cct7	Cys3, Ilv5, Stm1, Ura7, Vma2, Rnr4, Bmh1, Hsp60, Pdc1, Rpp0, Ssb1, Ssb2, Ura7, Rvb2	Rvb2 (Rvb1), Ssb1	Trr1, Oye2, Tsa1, Sti1, Hsc82, Kar2, Pre9, Sse2, Tps1, Gpd1, Hxk1, Gpm1, Rnr4	Kar2	Ssa1, Cad1, Rnr1, Rnr2, Oye3, Cct8, Gdh2, Pre3	Cad1, Cct8 (Cct7)

4.2. Metabolic regulation mediated MMS tolerance

The change in the growth curve was observed at the initial time of the MMS exposure (Fig. 1B). The diauxic growth may represent the shift in the metabolic pathway at the primary stage of MMS exposure. After some time the growth was rejuvenated again (Fig. 1B). It was noteworthy that 22 proteins that play role in metabolic pathways were up regulated in the MMS treatment (Supplementary Table 1). These proteins include ten glycolytic enzymes, nine proteins involved in biosynthesis of amino acids, four proteins involved in protein processing in endoplasmic reticulum, four proteins involved in RNA degradation and spliceosome, two in nitrogen metabolism and three in pentose phosphate pathway (Supplementary Table 1). Based on the image analysis, most of the identified proteins involved in carbon metabolism increased about 1.5–2 fold in the MMS treatment (Fig. 3B). In these proteins Adh1, Pdc1, Hxk2, Tdh2, Pgk1, Eno2, Eno1, Ilv5 and Hsc82 shows 2 fold or more increase in expression while Sse1 and Ssa1 shows the decrease in protein expression (Supplementary Table 1). The change in the expression of these genes probably leads to the shift in the growth curve. The PPP and pyruvate pathways showed greater change in expression as compared to the glycolysis pathway. This result indicates the shift from the glycolysis to the PPP pathway for carbohydrate metabolism which could lead to the generation of NADH required for cellular antioxidant systems [42].

4.3. MMS treatment induces antioxidative pathways

The metabolic response to DNA damage creates oxidative stress because of the generation of the MMS induced reactive oxygen species (ROS) [42]. Thioredoxin reductase (Trr1), alkyl hydroperoxide reductase (Ahp1) thioredoxin peroxidase (Tsa1) and superoxide dismutase (Sod1) are four main proteins involved in an enzymatic response signal against oxidative stress [43,44]. The results indicated the up-regulation of Trr1, Tsa1, and Ahp1 proteins. Trr1 is well recognized to destroy toxic radicals, while Tsa1 is a physiologically important antioxidant which is useful in an enzymatic defense against sulfur-containing radicals and provide protection against an oxidative system without thiol. In addition, the Ahp1 have role to reduce peroxides in oxidative stress condition [45]. These proteins have been also identified by Gasch et al after MMS treatment (Table 2). There are numerous reports of these proteins in stress response. Trabalzini et al and Zuzuarregui et al have presented experimental data on the induction of thioredoxin peroxidase at the late stages of fermentation [46,47]. Hirasawa et al has also reported a strong induction of this enzyme at the proteomic level in S. cerevisiae in response to high osmotic pressure conditions [48]. The altered metabolism contributes to suppression of the ROS while the antioxidative pathways participated in the scavenging of the ROS, both pathways improve the survivability of the cell under oxidative stress [[49], [50], [51]].

4.4. Oxidative stress induces CTD phosphorylation

In all eukaryotes, the transcription of mRNA is facilitated by the RNAP-II. The largest subunit of RNAP-II (Rpb1) contains a highly conserved C-terminal domain (CTD), which is composed of repeated unit of Y₁S₂P₃T₄S₅P₆S₇ consensus sequences. The CTD undergoes multiple posttranslational modifications which results in the recruitment and dissociation of a plethora of proteins to the CTD, essential for the active transcription [[12], [13], [14],28]. RNAP-II responds to DNA damage by increasing the CTD phosphorylation, especially Ser2 and factors that mediate or respond to this phosphorylation controlling the survival under DNA damage conditions [17,18]. The results indicated that the MMS treatment leads to changes in Ser2 as well as Ser5 and Ser7 CTD phosphorylation which lead to the hyper-phosphorylation of the CTD (Fig. 4A). The phosphorylation and dephosphorylation of CTD regulates the association of other transcriptional regulatory proteins to the RNAP-II as well as the transcription initiation, elongation, termination, mRNA-splicing and mRNA-export [16,17,52,53]. It means this epigenetic modification on the CTD regulates the global gene expression. This is why the MMS treatment leads to the up/down regulation of some metabolic pathways, oxidative pathway and DNA damage response pathway to protect the cell.

4.5. CTD phosphorylation leads to interactome change of RNAP-II

Although many of the proteins that comprise the initial response to MMS have been identified but, the complete repertoire of downstream DDR events induced by MMS especially with respect to the RNAPII remains poorly understood [[12], [13], [14], [15],54]. Comparison of fractions associated with RNAP-II in MMS treated sample indicate proteins that are differentially regulated and have a greater degree of association with RNAP-II in response to the MMS during active transcription. The whole cell proteomics approaches even after chromatin enrichment was able to identify very few proteins related to RNAP-II (Table 2, Kim et al., 2011). The approach using in solution digestion of proteins associated with RNAP-II and its subsequent analysis by nanoLC-MS/MS resulted in the identification of twenty seven RNAPII associated proteins after the 6 h of MMS exposure (Table 1). The network mapping of the RNAPII associated proteins using the string 10 showed that the identified proteins belong to the basal transcription factors and RNA biogenesis (Fig. 5). The MMS mediated hyperphosphorylation of the CTD led changing of the interactome which further govern the global gene regulation. In addition to these, the result identified the interaction of phosphatase (Rtr1) and kinase (Ire1) to the RNAP-II. Rtr1 is phosphatases which help in the dephosphorylation of the Ser5 position of the CTD which help in the transcription elongation and termination [55]. Irw1 was identified in the complex which involved in the transport of the RNAP-II complex to the nucleus where it further replaced by the transcription factors [56]. The Rtr1 have role in the CTD modification while the role of Ire1 in CTD modification is still unknown. The Ire1 have been identified alleviate endoplasmic reticulum stress by orchestrating the unfolded protein response [57]. The recruitment of these proteins might leads to the activation of the global transcription which may further regulate the variety of genes participates in oxidative stress produced by MMS.

5. Conclusion

The comparison of our proteome data with transcriptome data from the literature suggests that genes are regulated at different level; some are regulated at the transcriptomic level, whether some are regulated at proteomic level. The heat-shock proteins and chaperone proteins were significantly more abundant in the stress responses including oxidation and replication stress. To begin a characterization of proteins associated with active transcription in stress condition, we purified tagged RNAPII from S. cerevisiae. Several categories of transcription relevant proteins were co-purified. In the MMS treated condition we found approximately 27 proteins which were interacting with the complex. In case of treatment the phosphorylation status of the CTD changes and leads to increase in Ser2 phosphorylation, which may lead to the interaction of other proteins to RNAP-II. We have investigated the general protein composition in MMS stress condition in Saccharomyces cerevisiae which may leads to the identification of the potential targets and analysis of their role in cellular processes. This will not give only a global overview of the cellular changes elicited by stress, but also provide the framework for understanding the mechanisms behind MMS stress response in yeast.

Declaration of competing interest

The authors declare that they have no competing interests.

Author's contribution

APB participated in conceptualization, data curation, analysis, investigation, methodology, data validation and writing of the original draft. SK has done the formal analysis, validation of the data and writing the draft. MSA conceived the idea and participated in formal analysis and editing of the draft.

Funding

This work was supported by the Science and Engineering Research Board (SERB)-National Postdoctoral fellowship (DST No: PDF/2016/000937) and CSIR-CDRI. The grant provided for this study is primarily used for the design of the study, the collection, analysis of the data. Dr. Akhilendra Pratap Bharati is grateful to Indian Council of Agricultural Research and SERB (Department of Science and Technology) and Dr. Sohail Akhtar is grateful to Council of Scientific and Industrial Research-Central Drug Research Institute (CSIR-CDRI) for the financial assistance.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Appendix A. Supplementary data

The following is the Supplementary data to this article: