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Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

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Hypersensitivity of ppc1-537 to bleomycin that causes double-strand break (DSB). (a) The WT, ppc1-537 and Δrad3 mutant cells were grown at 26°C; spotted on the rich YPD medium containing DNA-damaging agents, phleomycin, hydroxyurea (HU) or camptothecin (CPT); irradiated by UV rays; and cultured at 26°C for 4 days. (b) Effect of bleomycin on ppc1-537 and Δrad3 was tested. Cells were grown exponentially at 26°C and then spotted at serial cell concentration on the YPD plates containing bleomycin (the concentration range, 0–8 mU ml−1), followed by incubation at 26°C or 30°C for 4 days. (c) Pulsed field gel electrophoresis (PFGE) of ppc1-537 mutant chromosome DNAs. PFGE analysis of S. pombe chromosomes was carried out. WT and ppc1-537 mutant cells were cultured at 26°C for 3 h after the addition of 0–10 mU ml−1 bleomycin to the medium. Schizosaccharomyces pombe has three chromosome DNAs that were degraded in the medium containing 3 or 10 mU ml−1 bleomycin. (d) Time course analysis of chromosome breakage by bleomycin was monitored by PFGE analysis. WT and ppc1-537 mutant cells were cultured at 26°C. Cells were harvested at indicated time points after addition of 3 mU ml−1 bleomycin. (e) The G0 cells of ppc1-537 were more sensitive to UV than the WT. See text.
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RSOB120117F5: Hypersensitivity of ppc1-537 to bleomycin that causes double-strand break (DSB). (a) The WT, ppc1-537 and Δrad3 mutant cells were grown at 26°C; spotted on the rich YPD medium containing DNA-damaging agents, phleomycin, hydroxyurea (HU) or camptothecin (CPT); irradiated by UV rays; and cultured at 26°C for 4 days. (b) Effect of bleomycin on ppc1-537 and Δrad3 was tested. Cells were grown exponentially at 26°C and then spotted at serial cell concentration on the YPD plates containing bleomycin (the concentration range, 0–8 mU ml−1), followed by incubation at 26°C or 30°C for 4 days. (c) Pulsed field gel electrophoresis (PFGE) of ppc1-537 mutant chromosome DNAs. PFGE analysis of S. pombe chromosomes was carried out. WT and ppc1-537 mutant cells were cultured at 26°C for 3 h after the addition of 0–10 mU ml−1 bleomycin to the medium. Schizosaccharomyces pombe has three chromosome DNAs that were degraded in the medium containing 3 or 10 mU ml−1 bleomycin. (d) Time course analysis of chromosome breakage by bleomycin was monitored by PFGE analysis. WT and ppc1-537 mutant cells were cultured at 26°C. Cells were harvested at indicated time points after addition of 3 mU ml−1 bleomycin. (e) The G0 cells of ppc1-537 were more sensitive to UV than the WT. See text.

Mentions: We found that ppc1-537 was hypersensitive to certain DNA-damaging agents at the permissive temperature. As shown in figure 5a, ppc1-537 was sensitive to 2.5 μg ml−1 DNA-breaking phleomycin at 26°C. It was moderately sensitive to 4 mM hydroxyurea (DNA replication inhibitor), but hardly sensitive to 5 µM camptothecin and to 50–100 J m−2 ultraviolet (UV) ray. The rad3 deletion (Rad3 is an ATR-like checkpoint kinase [45]) is the control strain for DNA damage sensitivity.Figure 5.


Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Hypersensitivity of ppc1-537 to bleomycin that causes double-strand break (DSB). (a) The WT, ppc1-537 and Δrad3 mutant cells were grown at 26°C; spotted on the rich YPD medium containing DNA-damaging agents, phleomycin, hydroxyurea (HU) or camptothecin (CPT); irradiated by UV rays; and cultured at 26°C for 4 days. (b) Effect of bleomycin on ppc1-537 and Δrad3 was tested. Cells were grown exponentially at 26°C and then spotted at serial cell concentration on the YPD plates containing bleomycin (the concentration range, 0–8 mU ml−1), followed by incubation at 26°C or 30°C for 4 days. (c) Pulsed field gel electrophoresis (PFGE) of ppc1-537 mutant chromosome DNAs. PFGE analysis of S. pombe chromosomes was carried out. WT and ppc1-537 mutant cells were cultured at 26°C for 3 h after the addition of 0–10 mU ml−1 bleomycin to the medium. Schizosaccharomyces pombe has three chromosome DNAs that were degraded in the medium containing 3 or 10 mU ml−1 bleomycin. (d) Time course analysis of chromosome breakage by bleomycin was monitored by PFGE analysis. WT and ppc1-537 mutant cells were cultured at 26°C. Cells were harvested at indicated time points after addition of 3 mU ml−1 bleomycin. (e) The G0 cells of ppc1-537 were more sensitive to UV than the WT. See text.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3472395&req=5

RSOB120117F5: Hypersensitivity of ppc1-537 to bleomycin that causes double-strand break (DSB). (a) The WT, ppc1-537 and Δrad3 mutant cells were grown at 26°C; spotted on the rich YPD medium containing DNA-damaging agents, phleomycin, hydroxyurea (HU) or camptothecin (CPT); irradiated by UV rays; and cultured at 26°C for 4 days. (b) Effect of bleomycin on ppc1-537 and Δrad3 was tested. Cells were grown exponentially at 26°C and then spotted at serial cell concentration on the YPD plates containing bleomycin (the concentration range, 0–8 mU ml−1), followed by incubation at 26°C or 30°C for 4 days. (c) Pulsed field gel electrophoresis (PFGE) of ppc1-537 mutant chromosome DNAs. PFGE analysis of S. pombe chromosomes was carried out. WT and ppc1-537 mutant cells were cultured at 26°C for 3 h after the addition of 0–10 mU ml−1 bleomycin to the medium. Schizosaccharomyces pombe has three chromosome DNAs that were degraded in the medium containing 3 or 10 mU ml−1 bleomycin. (d) Time course analysis of chromosome breakage by bleomycin was monitored by PFGE analysis. WT and ppc1-537 mutant cells were cultured at 26°C. Cells were harvested at indicated time points after addition of 3 mU ml−1 bleomycin. (e) The G0 cells of ppc1-537 were more sensitive to UV than the WT. See text.
Mentions: We found that ppc1-537 was hypersensitive to certain DNA-damaging agents at the permissive temperature. As shown in figure 5a, ppc1-537 was sensitive to 2.5 μg ml−1 DNA-breaking phleomycin at 26°C. It was moderately sensitive to 4 mM hydroxyurea (DNA replication inhibitor), but hardly sensitive to 5 µM camptothecin and to 50–100 J m−2 ultraviolet (UV) ray. The rad3 deletion (Rad3 is an ATR-like checkpoint kinase [45]) is the control strain for DNA damage sensitivity.Figure 5.

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

Show MeSH
Related in: MedlinePlus