Altered S-AdenosylMethionine availability impacts dNTP pools in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.

Keywords: AdoMet synthetases; Saccharomyces cerevisiae; S‐adenosylmethionine (AdoMet); genomic instability; methyl cycle; yeast genetics.

FIGURE 1

Schematic illustration of the methyl cycle and interconnected pathways. Sam1 and Sam2 function in the methyl cycle (brown) to produce S-AdenosylMethionine (AdoMet), the main methyl donor in cells, from Methionine (Met) and ATP. Products of the methyl cycle are used in three main pathways, aminopropylation (purple), transmethylation (blue), and transsulfuration (green). The folate cycle (pink) is also directly connected to the methyl cycle, providing an alternate methyl donor for the conversion of Homocysteine (Hcy) to Met. AdoMet is used directly in synthesis of polyamines, spermine and spermidine, as part of aminopropylation. AdoMet decarboxylase forms decarboxylated-AdoMet (dc-AdoMet), from AdoMet, which can then be used as an aminopropyl group donor in spermidine or spermine synthesis. When not used in aminopropylation, AdoMet-Dependent MethylTransferases (ADMTs) transfer a methyl group from AdoMet to a recipient molecule (RNA, lipids, proteins, and small molecules in S. cerevisiae), leaving S-AdenosylHomocysteine (AdoHcy), constituting the transmethylation reactions. AdenosylHomoCYsteinase (AHCY) can then act on AdoHcy to produce Hcy. Next, within the methyl cycle, Homocysteine MethylTransferase (HMT) can convert Hcy into Met using 5-Methyl-TetraHydroFolate vitamers (5-MTHF) from the folate cycle as the methyl donor, where TetraHydroFolate (THF) is the byproduct of this process. The folate cycle is made up of interconversions between many folate vitamers and is important in pyrimidine synthesis where Cdc21 uses 5,10-methylene-THF (5,10-MTHF) as a methyl donor for the conversion of dUMP to dTMP. 5,10-MTHF can also be converted to 10-formyl-THF and used in purine synthesis. Conversely, Hcy can be shuttled into transsulfuration processes where it is converted into cystathionine then cysteine using cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) enzymes, respectively. Cysteine can then be converted to γ-glutamyl-l-cysteinyl-glycine (GSH). In the presence of reactive oxygen species (ROS), Glutathione Transferases (GST) and Glutaredoxins (Grx) use reduced GSH to neutralize ROS toxicity, with GSH-S-conjugates and oxidized GSH (GSSG) formed in the process, respectively. The recycling of GSSG to GSH is catalyzed by glutathione reductase (GR) which utilizes NADPH as the electron donor. NADPH can be regenerated in many reactions, including by glucose-6-phosphate dehydrogenase (G6PD) and NADP-isocitrate dehydrogenase (NADP), both of which are activated by spermine and spermidine. Arrows indicate reaction conversions, whereas dashed lines indicated activation steps.

FIGURE 2

Quantification of cellular dNTP levels in wildtype, sam1Δ/sam1Δ and sam2Δ/sam2Δ S. cerevisiae. The levels of dATP, dTTP, dCTP, and dGTP were investigated via a fluorescence-based qPCR methodology. Fold change differences were calculated as compared to wildtype. Results for the sam1Δ/sam1Δ strain are shown via the orange bars and the sam2Δ/sam2Δ strain via the dark gray bar. Data represent the mean of at least three separate experiments and error bars represent ± SD. (a) The fold change of individual dNTPs from both sam1Δ/sam1Δ and sam2Δ/sam2Δ strains compared to wildtype. (b) The fold change of the overall dNTP pool from both sam1Δ/sam1Δ and sam2Δ/sam2Δ strains compared to wildtype. An (*) represents a statistic difference of p≤ 0.05. A dashed line is drawn at 1 for comparison of the fold change differences from wildtype levels. qPCR, quantitative polymerase chain reaction; SD, standard deviation.

FIGURE 3

Comparison of endogenous ATP levels in our mutant strains. Endogenous levels of ATP were detected in exponential phase cells via a luciferin/luciferase kit. The level of ATP was derived from a generated standard curve. Data represent the mean of at least three separate experiments and error bars represent ± SD. Results for the wildtype strain are shown via the blue bar, the sam1Δ/sam1Δ strain is shown via the orange bar and the sam2Δ/sam2Δ strain via the dark gray bar. An (*) represents a statistic difference of p ≤ 0.05. SD, standard deviation.

FIGURE 4

Determination of total glutathione, GSSG, and reduced GSH. Supernatants and masked supernatants from each strain were used to assay total glutathione and GSSG. A standard curve was generated and used to convert absorbance data into concentration. The BCA method (Pierce^® BCA Protein assay kit) was used to determine the concentration of total protein. Results are shown in μM/mg protein ± SD and represent the mean of at least three separate experiments. Results for the wildtype strain are shown via the blue bars, the sam1Δ/sam1Δ strain is shown via the orange bars and the sam2Δ/sam2Δ strain via the dark gray bars. (a) The total glutathione level of each strain. (b) The GSH level in each strain was calculated from total glutathione [(GSH + GSSG) − GSSG × 2]. (c) The GSSG level of each strain. An (*) represents a statistic difference of p ≤ 0.05. BCA, bicinchoninic acid; GSH, g-glutamyl-l-cysteinyl-glycine; GSSG, oxidized glutathione; SD, standard deviation.

FIGURE 5

Glutathione S-transferase (GST) activity in wildtype, sam1Δ/sam1Δ and sam2Δ/sam2Δ S. cerevisiae strains. The GST level of each sample was determined from extracted supernatant based on the interaction with 1-Chloro-2,4-dinitrobenzene (CDNB). The protein concentration was determined following the BCA method (Pierce^® BCA Protein assay kit). GST enzyme activity was determined by monitoring the changes in absorbance at 340 nm and reported as nmol/min/mg protein. Data represent the mean of at least three separate experiments and error bars represent ± SD. Results for the wildtype strain are shown via the blue bar, the sam1Δ/sam1Δ strain is shown via the orange bar and the sam2Δ/sam2Δ strain via the dark gray bar. BCA, bicinchoninic acid; SD, standard deviation.

FIGURE 6

Fold change of reactive oxygen species (ROS) levels due to loss of the SAM1 and SAM2 genes. The level of ROS was determined using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA). The supernatant of each sample was measured at the excitation (504 nm (524 nm) wavelengths for H₂DCF-DA. The ROS content is reported as relative fluorescence units. Data represent the mean of at least three separate experiments and error bars represent ± SD. Results for the wildtype strain are shown via the blue bar, the sam1Δ/sam1Δ strain is shown via the orange bar and the sam2Δ/sam2Δ strain via the dark gray bar. SD, standard deviation.

FIGURE 7

Polyamine levels in in wildtype, sam1Δ/sam1Δ and sam2Δ/sam2Δ strains. Samples from each strain were measured to detect the fluorescence of total polyamines at the excitation of 535 nm and emission of 587 nm. A standard curve was generated and used to convert fluorescent units to nmol of total polyamines. The total protein concentration was determined using the BCA method (Pierce^® BCA Protein assay kit). The nmol of total polyamines/mg protein was calculated and data represent the mean of at least three separate experiments and error bars represent ± SD. Results for the wildtype strain are shown via the blue bar, the sam1Δ/sam1Δ strain is shown via the orange bar and the sam2Δ/sam2Δ strain via the dark gray bar. BCA, bicinchoninic acid; SD, standard deviation.