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A screen for suppressors of gross chromosomal rearrangements identifies a conserved role for PLP in preventing DNA lesions.

Kanellis P, Gagliardi M, Banath JP, Szilard RK, Nakada S, Galicia S, Sweeney FD, Cabelof DC, Olive PL, Durocher D - PLoS Genet. (2007)

Bottom Line: We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint.Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase.Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

ABSTRACT
Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

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PLP levels Are Required for Optimal dTMP Biosynthesis(A) The intersection of vitamin B6 and dTMP biosynthesis. (i) Dietary vitamin B6 (i.e., pyridoxine) is imported into the cell via the Tpn1 transporter (S. cerevisiae gene names in brackets). (ii) Pdxk phosphorylates the B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) to generate PLP, which acts (iii) as a cofactor for serine hydroxymethyl transferase (SHMT). (iv) SHMT is necessary for the formation of methylenetetrahydrofolate (CH2=THF), the methyl group donor for the conversion of dUMP into dTMP. (v) A deficiency in PLP is predicted to reduce dTMP levels leading to a nucleotide pool imbalance and incorporation of uracil into DNA.(B) bud16Δ cells accumulate uracil in DNA, as measured by a modified aldehydic slot blot assay. The results of the slot blot (bottom panel) were quantified and shown in the graph. IDV refers to the integrated density values of the bands.(C) Uracil accumulation in the bud16Δ genomic DNA is comparable to that observed in ung1Δ DNA.(D) bud16Δ cells are sensitive to nucleotide depletion by hydroxyurea at 0.2 M. The results of a colony forming assay can also be found in Table S3.
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pgen-0030134-g008: PLP levels Are Required for Optimal dTMP Biosynthesis(A) The intersection of vitamin B6 and dTMP biosynthesis. (i) Dietary vitamin B6 (i.e., pyridoxine) is imported into the cell via the Tpn1 transporter (S. cerevisiae gene names in brackets). (ii) Pdxk phosphorylates the B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) to generate PLP, which acts (iii) as a cofactor for serine hydroxymethyl transferase (SHMT). (iv) SHMT is necessary for the formation of methylenetetrahydrofolate (CH2=THF), the methyl group donor for the conversion of dUMP into dTMP. (v) A deficiency in PLP is predicted to reduce dTMP levels leading to a nucleotide pool imbalance and incorporation of uracil into DNA.(B) bud16Δ cells accumulate uracil in DNA, as measured by a modified aldehydic slot blot assay. The results of the slot blot (bottom panel) were quantified and shown in the graph. IDV refers to the integrated density values of the bands.(C) Uracil accumulation in the bud16Δ genomic DNA is comparable to that observed in ung1Δ DNA.(D) bud16Δ cells are sensitive to nucleotide depletion by hydroxyurea at 0.2 M. The results of a colony forming assay can also be found in Table S3.

Mentions: We finally sought to narrow down the biological pathway in which PLP acts to promote genome stability. This is a difficult task, since PLP is a critical cofactor for numerous essential enzymes acting in amino acid and dTMP biosynthesis. However, our observations in yeast and human cells indicate a role for PLP in preventing DNA lesions during DNA replication, pointing to dTMP synthesis as the likeliest candidate pathway linking PLP to genome stability (Figure 8A). This possible association is strengthened by the multitude of observations that link dTMP biosynthesis to genome integrity in both prokaryotes and eukaryotes (reviewed in [47]). In this context, PLP deficiency may either cause aberrant uracil incorporation into DNA, generate a nucleotide imbalance that impairs DNA replication fork stability, or both. We therefore sought to assess the involvement of PLP in dTMP biosynthesis by testing whether bud16Δ cells accumulate uracil nucleotides in their DNA. To do so, we employed a recently described modified aldehydic slot blot assay that detects abasic sites produced when isolated DNA is treated with a uracil glycosylase enzyme [48]. As shown in Figure 8B, strains lacking Pdxk (bud16Δ) accumulate uracil in their genome significantly more than their wild-type counterparts. This accumulation is likely to be biologically important, as it is in the same range as the uracil accumulation observed in cells deficient in uracil glycosylase (ung1Δ cells), the main enzyme dedicated to the removal of uracil in DNA (Figure 8C). Furthermore, the double ung1Δ bud16Δ mutant accumulates more uracil in its genome than either of the single mutants, suggesting that UNG1 and BUD16 function in separate pathways to prevent uracil incorporation into DNA. These results are therefore consistent with a model in which the bud16Δ mutation increases dUMP pools, thereby increasing the frequency of dUTP incorporation into DNA.


A screen for suppressors of gross chromosomal rearrangements identifies a conserved role for PLP in preventing DNA lesions.

Kanellis P, Gagliardi M, Banath JP, Szilard RK, Nakada S, Galicia S, Sweeney FD, Cabelof DC, Olive PL, Durocher D - PLoS Genet. (2007)

PLP levels Are Required for Optimal dTMP Biosynthesis(A) The intersection of vitamin B6 and dTMP biosynthesis. (i) Dietary vitamin B6 (i.e., pyridoxine) is imported into the cell via the Tpn1 transporter (S. cerevisiae gene names in brackets). (ii) Pdxk phosphorylates the B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) to generate PLP, which acts (iii) as a cofactor for serine hydroxymethyl transferase (SHMT). (iv) SHMT is necessary for the formation of methylenetetrahydrofolate (CH2=THF), the methyl group donor for the conversion of dUMP into dTMP. (v) A deficiency in PLP is predicted to reduce dTMP levels leading to a nucleotide pool imbalance and incorporation of uracil into DNA.(B) bud16Δ cells accumulate uracil in DNA, as measured by a modified aldehydic slot blot assay. The results of the slot blot (bottom panel) were quantified and shown in the graph. IDV refers to the integrated density values of the bands.(C) Uracil accumulation in the bud16Δ genomic DNA is comparable to that observed in ung1Δ DNA.(D) bud16Δ cells are sensitive to nucleotide depletion by hydroxyurea at 0.2 M. The results of a colony forming assay can also be found in Table S3.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1941753&req=5

pgen-0030134-g008: PLP levels Are Required for Optimal dTMP Biosynthesis(A) The intersection of vitamin B6 and dTMP biosynthesis. (i) Dietary vitamin B6 (i.e., pyridoxine) is imported into the cell via the Tpn1 transporter (S. cerevisiae gene names in brackets). (ii) Pdxk phosphorylates the B6 vitamers (pyridoxine, pyridoxamine, and pyridoxal) to generate PLP, which acts (iii) as a cofactor for serine hydroxymethyl transferase (SHMT). (iv) SHMT is necessary for the formation of methylenetetrahydrofolate (CH2=THF), the methyl group donor for the conversion of dUMP into dTMP. (v) A deficiency in PLP is predicted to reduce dTMP levels leading to a nucleotide pool imbalance and incorporation of uracil into DNA.(B) bud16Δ cells accumulate uracil in DNA, as measured by a modified aldehydic slot blot assay. The results of the slot blot (bottom panel) were quantified and shown in the graph. IDV refers to the integrated density values of the bands.(C) Uracil accumulation in the bud16Δ genomic DNA is comparable to that observed in ung1Δ DNA.(D) bud16Δ cells are sensitive to nucleotide depletion by hydroxyurea at 0.2 M. The results of a colony forming assay can also be found in Table S3.
Mentions: We finally sought to narrow down the biological pathway in which PLP acts to promote genome stability. This is a difficult task, since PLP is a critical cofactor for numerous essential enzymes acting in amino acid and dTMP biosynthesis. However, our observations in yeast and human cells indicate a role for PLP in preventing DNA lesions during DNA replication, pointing to dTMP synthesis as the likeliest candidate pathway linking PLP to genome stability (Figure 8A). This possible association is strengthened by the multitude of observations that link dTMP biosynthesis to genome integrity in both prokaryotes and eukaryotes (reviewed in [47]). In this context, PLP deficiency may either cause aberrant uracil incorporation into DNA, generate a nucleotide imbalance that impairs DNA replication fork stability, or both. We therefore sought to assess the involvement of PLP in dTMP biosynthesis by testing whether bud16Δ cells accumulate uracil nucleotides in their DNA. To do so, we employed a recently described modified aldehydic slot blot assay that detects abasic sites produced when isolated DNA is treated with a uracil glycosylase enzyme [48]. As shown in Figure 8B, strains lacking Pdxk (bud16Δ) accumulate uracil in their genome significantly more than their wild-type counterparts. This accumulation is likely to be biologically important, as it is in the same range as the uracil accumulation observed in cells deficient in uracil glycosylase (ung1Δ cells), the main enzyme dedicated to the removal of uracil in DNA (Figure 8C). Furthermore, the double ung1Δ bud16Δ mutant accumulates more uracil in its genome than either of the single mutants, suggesting that UNG1 and BUD16 function in separate pathways to prevent uracil incorporation into DNA. These results are therefore consistent with a model in which the bud16Δ mutation increases dUMP pools, thereby increasing the frequency of dUTP incorporation into DNA.

Bottom Line: We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint.Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase.Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

ABSTRACT
Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

Show MeSH
Related in: MedlinePlus