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Tryptophan-Dependent Control of Colony Formation After DNA Damage via Sea3-Regulated TORC1 Signaling in Saccharomyces cerevisiae.

Polleys EJ, Bertuch AA - G3 (Bethesda) (2015)

Bottom Line: High levels of tryptophan in yeast peptone dextrose media did not rescue the delay in colony formation, suggesting a defect in tryptophan import, although levels of the high-affinity tryptophan permease Tat2 were not perturbed in the sea3Δ mutant.Addition of quinolinic acid, an intermediate of the de novo NAD+ biosynthetic pathway, however, rescued the delay in colony formation in the sea3Δ mutant.Together, these findings highlight the importance of enforcement of TORC1 signaling and suggest that internal tryptophan levels influence growth recovery post DNA damage through the role of tryptophan in NAD+ synthesis.

View Article: PubMed Central - PubMed

Affiliation: Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas 77030.

No MeSH data available.


Related in: MedlinePlus

Expression of TRP1, but not exogenous tryptophan, rescues the delay in colony formation in sea3Δ mutants. (A) Fivefold serial dilutions of wild-type and sea3Δ mutants in the break-induced replication (BIR) assay strain with and without the addition of the TRP1 plasmid pRS414 on YPD and YPGal. The wild-type-in and sea3∆::LEU2-in strains have been previously experienced HO-induction with galactose and undergone a repair event, rendering them unable to undergo HO-mediated DSB induction upon replating on galactose. (B) Fivefold serial dilutions of the wild-type and sea3Δ BIR assay strain mutants plated on YPD, YPGal, YPD + 100 μM tryptophan (Trp) and YPGal + 100 μM Trp. (C) Western blots showing Tat2-3XFlag levels post-galactose induction at the indicated time points. Whole-cell extracts were prepared and blotted with α-Flag. Images are representative of three independent experiments. (D) Fivefold serial dilutions of the wild-type and sea3Δ mutants in the BIR assay strain were plated on YPD, YPGal and YPD and YPGal with either 2 or 4 μM quinolinic acid (QA) added. Plates were imaged on day 3, day 4, and day 5.
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fig5: Expression of TRP1, but not exogenous tryptophan, rescues the delay in colony formation in sea3Δ mutants. (A) Fivefold serial dilutions of wild-type and sea3Δ mutants in the break-induced replication (BIR) assay strain with and without the addition of the TRP1 plasmid pRS414 on YPD and YPGal. The wild-type-in and sea3∆::LEU2-in strains have been previously experienced HO-induction with galactose and undergone a repair event, rendering them unable to undergo HO-mediated DSB induction upon replating on galactose. (B) Fivefold serial dilutions of the wild-type and sea3Δ BIR assay strain mutants plated on YPD, YPGal, YPD + 100 μM tryptophan (Trp) and YPGal + 100 μM Trp. (C) Western blots showing Tat2-3XFlag levels post-galactose induction at the indicated time points. Whole-cell extracts were prepared and blotted with α-Flag. Images are representative of three independent experiments. (D) Fivefold serial dilutions of the wild-type and sea3Δ mutants in the BIR assay strain were plated on YPD, YPGal and YPD and YPGal with either 2 or 4 μM quinolinic acid (QA) added. Plates were imaged on day 3, day 4, and day 5.

Mentions: To determine whether the growth delay was caused by a general sensitivity to galactose (rather than the DSB induced by galactose), we took sea3Δ mutants in the BIR strain background that had been plated previously on galactose, had undergone BIR repair of the DSB and, therefore, would not sustain another HO-induced DSB when replated on galactose, and compared their growth on galactose to sea3Δ mutants that had not been previously exposed to galactose and, therefore, would undergo DSB induction when plated on galactose. If the growth delay of the sea3Δ mutants on galactose were simply due to a general sensitivity to galactose, then the presence or absence of a cleavable HO cut site would not matter and both types of sea3Δ mutants would be equally sensitive to galactose. We found, however, that when the sea3Δ mutants that were originally plated on galactose were re-plated on galactose, quantifiable colonies appeared sooner (Figure S2; see also Figure 5A and Figure S8A), suggesting that the delay in colony formation in the sea3Δ mutant was not simply due to a sensitivity to galactose.


Tryptophan-Dependent Control of Colony Formation After DNA Damage via Sea3-Regulated TORC1 Signaling in Saccharomyces cerevisiae.

Polleys EJ, Bertuch AA - G3 (Bethesda) (2015)

Expression of TRP1, but not exogenous tryptophan, rescues the delay in colony formation in sea3Δ mutants. (A) Fivefold serial dilutions of wild-type and sea3Δ mutants in the break-induced replication (BIR) assay strain with and without the addition of the TRP1 plasmid pRS414 on YPD and YPGal. The wild-type-in and sea3∆::LEU2-in strains have been previously experienced HO-induction with galactose and undergone a repair event, rendering them unable to undergo HO-mediated DSB induction upon replating on galactose. (B) Fivefold serial dilutions of the wild-type and sea3Δ BIR assay strain mutants plated on YPD, YPGal, YPD + 100 μM tryptophan (Trp) and YPGal + 100 μM Trp. (C) Western blots showing Tat2-3XFlag levels post-galactose induction at the indicated time points. Whole-cell extracts were prepared and blotted with α-Flag. Images are representative of three independent experiments. (D) Fivefold serial dilutions of the wild-type and sea3Δ mutants in the BIR assay strain were plated on YPD, YPGal and YPD and YPGal with either 2 or 4 μM quinolinic acid (QA) added. Plates were imaged on day 3, day 4, and day 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4502372&req=5

fig5: Expression of TRP1, but not exogenous tryptophan, rescues the delay in colony formation in sea3Δ mutants. (A) Fivefold serial dilutions of wild-type and sea3Δ mutants in the break-induced replication (BIR) assay strain with and without the addition of the TRP1 plasmid pRS414 on YPD and YPGal. The wild-type-in and sea3∆::LEU2-in strains have been previously experienced HO-induction with galactose and undergone a repair event, rendering them unable to undergo HO-mediated DSB induction upon replating on galactose. (B) Fivefold serial dilutions of the wild-type and sea3Δ BIR assay strain mutants plated on YPD, YPGal, YPD + 100 μM tryptophan (Trp) and YPGal + 100 μM Trp. (C) Western blots showing Tat2-3XFlag levels post-galactose induction at the indicated time points. Whole-cell extracts were prepared and blotted with α-Flag. Images are representative of three independent experiments. (D) Fivefold serial dilutions of the wild-type and sea3Δ mutants in the BIR assay strain were plated on YPD, YPGal and YPD and YPGal with either 2 or 4 μM quinolinic acid (QA) added. Plates were imaged on day 3, day 4, and day 5.
Mentions: To determine whether the growth delay was caused by a general sensitivity to galactose (rather than the DSB induced by galactose), we took sea3Δ mutants in the BIR strain background that had been plated previously on galactose, had undergone BIR repair of the DSB and, therefore, would not sustain another HO-induced DSB when replated on galactose, and compared their growth on galactose to sea3Δ mutants that had not been previously exposed to galactose and, therefore, would undergo DSB induction when plated on galactose. If the growth delay of the sea3Δ mutants on galactose were simply due to a general sensitivity to galactose, then the presence or absence of a cleavable HO cut site would not matter and both types of sea3Δ mutants would be equally sensitive to galactose. We found, however, that when the sea3Δ mutants that were originally plated on galactose were re-plated on galactose, quantifiable colonies appeared sooner (Figure S2; see also Figure 5A and Figure S8A), suggesting that the delay in colony formation in the sea3Δ mutant was not simply due to a sensitivity to galactose.

Bottom Line: High levels of tryptophan in yeast peptone dextrose media did not rescue the delay in colony formation, suggesting a defect in tryptophan import, although levels of the high-affinity tryptophan permease Tat2 were not perturbed in the sea3Δ mutant.Addition of quinolinic acid, an intermediate of the de novo NAD+ biosynthetic pathway, however, rescued the delay in colony formation in the sea3Δ mutant.Together, these findings highlight the importance of enforcement of TORC1 signaling and suggest that internal tryptophan levels influence growth recovery post DNA damage through the role of tryptophan in NAD+ synthesis.

View Article: PubMed Central - PubMed

Affiliation: Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas 77030.

No MeSH data available.


Related in: MedlinePlus