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. 2016 May;33(5):183-9.
doi: 10.1002/yea.3150. Epub 2016 Mar 21.

Tryptophan Biosynthesis Is Important for Resistance to Replicative Stress in Saccharomyces Cerevisiae

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Free PMC article

Tryptophan Biosynthesis Is Important for Resistance to Replicative Stress in Saccharomyces Cerevisiae

Stephen K Godin et al. Yeast. .
Free PMC article

Abstract

Acute tryptophan depletion is used to induce low levels of serotonin in the brain. This method has been widely used in psychiatric studies to evaluate the effect of low levels of serotonin, and is generally considered a safe and reversible procedure. Here we use the budding yeast Saccharomyces cerevisiae to study the effects of tryptophan depletion on growth rate upon exposure to DNA-damaging agents. Surprisingly, we found that budding yeast undergoing tryptophan depletion were more sensitive to DNA-damaging agents such as methyl methanesulphonate (MMS) and hydroxyurea (HU). We found that this defect was independent of several DNA repair pathways, such as homologous recombination, base excision repair and translesion synthesis, and that this damage sensitivity was not due to impaired S-phase signalling. Upon further analysis, we found that the DNA-damage sensitivity of tryptophan depletion was likely due to impaired protein synthesis. These studies describe an important source of variance in budding yeast when using tryptophan as an auxotrophic marker, particularly on studies focusing on DNA repair, and suggest that further testing of the effect of tryptophan depletion on DNA repair in mammalian cells is warranted. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: DNA repair; DNA replication; Protein synthesis; Saccharomyces; Tryptophan.

Conflict of interest statement

Conflict of interest statement. None declared.

Figures

FIGURE 1
FIGURE 1
trp1-1 W303 cells are sensitive to MMS and HU. A. The indicated strains were five-fold serially diluted onto YPD medium or YPD containing 0.02% MMS or 100mM HU and incubated for 2 days at 30°C or four days at 23°C. B. TRP1 and trp1-1 cells or trp1-1 cells containing the indicated plasmid were five-fold serially diluted onto YPD medium or YPD containing 0.02% MMS or 100mM HU and incubated for two days at 30°C or four days at 23°C. C. The indicated strains were five-fold serially diluted onto SC medium or SC containing 0.02% MMS or 100mM HU and incubated for 2 days at 30°C or four days at 23°C.
FIGURE 2
FIGURE 2
The defect caused by trp1-1 is observed in multiple yeast strain backgrounds and is observed with deletion of any tryptophan biosynthesis gene in the BY strain background. A. TRP1 and trp1Δ cells containing the indicated plasmid are five-fold serially diluted onto YPD medium or YPD containing 100mM HU and incubated for two days at 30°C or four days at 23°C. B. TRP+, trp1Δ, trp2Δ, trp3Δ, trp4Δ, or trp5Δ cells are five fold serially diluted and plated onto YPD, YPD with 0.02% MMS, or YPD with 100mM HU as in A. C. TRP+, trp1Δ, trp2Δ, trp3Δ, trp4Δ, or trp5Δ cells are five fold serially diluted and plated onto SC, SC with 0.02% MMS, SC with 100mM HU as in B. Note that the tryptophan mutants, while always more sensitive than TRP+ cells to medium containing MMS or HU, exhibited variable growth between trials.
FIGURE 3
FIGURE 3
trp1-1 cells MMS sensitivity is independent of the major DNA repair pathways in W303 yeast. A. WT, rev3Δ, mag1Δ, and rad57Δ cells that are TRP1 or trp1-1 were grown overnight at 23°C and five-fold serially diluted onto YPD medium or YPD with 0.006% MMS and incubated for two days at 30°C or four days at 23°C. B. TRP1 and trp1-1 cells were grown at 30°C or 23°C in SC or SC containing 0.0005% MMS to measure recombination rates as described for the direct repeat recombination assay(Godin, et al., 2013). C. WT, tof1Δ, rad9Δ, and mrc1Δ cells that are TRP1 or trp1-1 were grown overnight at 23°C and diluted as in A.
FIGURE 4
FIGURE 4
The MMS sensitivity of a trp1-1 cell is not due to defects in NAD+ metabolism in W303 yeast but is due to slowed protein synthesis. A. WT, bna2Δ, and sir2Δ cells that are TRP1 or trp1-1 are grown overnight at 23°C and five-fold serially diluted onto YPD medium or YPD with 0.012% MMS and incubated for two days at 30°C or four days at 23°C. B. WT (TRP1) and trp1-1 cells were grown overnight at 30°C and five-fold serially diluted onto rich YPD medium + 0.03% DMSO or YPD medium + 0.03% DMSO containing 100 ng/mL cycloheximide (CHX) either with or without 0.012% MMS. The plates were incubated at 23°C for four days or 30°C for three days prior to imaging.

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