Limits...
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.

Show MeSH

Related in: MedlinePlus

The ChrXV-L GCR Assay(A) Schematic of the ChrXV-L GCR reporter chromosome. A GCR event in this region of ChrXV-L can result in the loss of the URA3 and CAN1 genes, which yields a canavanine and 5-FOA resistant strain (canR 5-FOAR). PSF3 is the first essential gene on ChrXV-L. HRI and HRII denote two regions of homology that are centered around the PAU20 and HXT11 genes, respectively.(B) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the parent strain (W) or strains that have undergone a GCR event (1–9). Asterisk indicates incomplete digestion products of ChrXV.(C) Array-based comparative genome hybridization of two strains that have undergone a GCR event at ChrXV-L. The above panel is a histogram representation of log2-tranformed relative signal enrichments on Chromosome XV viewed in the University of California Santa Cruz genome browser [69]. The location of the array probes (reporters) is also indicated. Note the large loss of sequences on the left arm of ChrXV in both strains. The lower panel zooms to the ChrXV-L subtelomeric region. The breakpoint in strain (i) must be in the vicinity of the PAU20 gene (YOL161C), whereas that of strain (ii) resides in the vicinity of HXT11.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1941753&req=5

pgen-0030134-g001: The ChrXV-L GCR Assay(A) Schematic of the ChrXV-L GCR reporter chromosome. A GCR event in this region of ChrXV-L can result in the loss of the URA3 and CAN1 genes, which yields a canavanine and 5-FOA resistant strain (canR 5-FOAR). PSF3 is the first essential gene on ChrXV-L. HRI and HRII denote two regions of homology that are centered around the PAU20 and HXT11 genes, respectively.(B) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the parent strain (W) or strains that have undergone a GCR event (1–9). Asterisk indicates incomplete digestion products of ChrXV.(C) Array-based comparative genome hybridization of two strains that have undergone a GCR event at ChrXV-L. The above panel is a histogram representation of log2-tranformed relative signal enrichments on Chromosome XV viewed in the University of California Santa Cruz genome browser [69]. The location of the array probes (reporters) is also indicated. Note the large loss of sequences on the left arm of ChrXV in both strains. The lower panel zooms to the ChrXV-L subtelomeric region. The breakpoint in strain (i) must be in the vicinity of the PAU20 gene (YOL161C), whereas that of strain (ii) resides in the vicinity of HXT11.

Mentions: Most of the current knowledge regarding GCR formation originates from candidate gene studies examining rearrangements at a single locus in budding yeast, the left arm of Chromosome V (ChrV-L). Although this locus has been instrumental in the deciphering of many basic mechanisms governing genome stability in eukaryotes, examination of GCR formation at other loci provides a complementary view. For example, the use of yeast artificial chromosomes to study GCRs led to the discovery that defective chromosome condensation (in a ycs4 mutant) results in GCR events [7]. In addition, studies employing a Chromosome VII disome found that defects in DNA replication and checkpoint control elevate rates of chromosome loss and rearrangements following replication fork stalling [11]. In another study, Hackett et al. employed the telomeric region of ChrXV-L to study GCR events triggered by telomerase dysfunction [12]. This latter locus is particularly useful since GCRs at ChrXV-L involve break-induced replication (BIR), a type of homologous recombination repair predicted to be a major source of genome rearrangements [2,13–15]. In contrast, GCRs formed at ChrV-L are primarily the consequence of de novo telomere addition [8]. This difference can be explained by the architecture of the telomere-proximal region on ChrXV-L, which contains two regions of homology (HRI centered on the PAU20 gene, and HRII centered on the HXT11 gene; Figure 1A) located 12 kb and 25 kb from the telomere [12]. These regions share a high degree of sequence identity with other regions in the genome [12]. As a consequence, DNA lesions formed at loci telomeric to HRI or HRII are predominantly repaired by BIR, producing nonreciprocal translocations in haploid cells. Notably, increased repair by BIR can also lead to loss of heterozygosity in diploid genomes, which may accelerate the process of tumorigenesis by inactivation of tumor suppressor genes.


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)

The ChrXV-L GCR Assay(A) Schematic of the ChrXV-L GCR reporter chromosome. A GCR event in this region of ChrXV-L can result in the loss of the URA3 and CAN1 genes, which yields a canavanine and 5-FOA resistant strain (canR 5-FOAR). PSF3 is the first essential gene on ChrXV-L. HRI and HRII denote two regions of homology that are centered around the PAU20 and HXT11 genes, respectively.(B) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the parent strain (W) or strains that have undergone a GCR event (1–9). Asterisk indicates incomplete digestion products of ChrXV.(C) Array-based comparative genome hybridization of two strains that have undergone a GCR event at ChrXV-L. The above panel is a histogram representation of log2-tranformed relative signal enrichments on Chromosome XV viewed in the University of California Santa Cruz genome browser [69]. The location of the array probes (reporters) is also indicated. Note the large loss of sequences on the left arm of ChrXV in both strains. The lower panel zooms to the ChrXV-L subtelomeric region. The breakpoint in strain (i) must be in the vicinity of the PAU20 gene (YOL161C), whereas that of strain (ii) resides in the vicinity of HXT11.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0030134-g001: The ChrXV-L GCR Assay(A) Schematic of the ChrXV-L GCR reporter chromosome. A GCR event in this region of ChrXV-L can result in the loss of the URA3 and CAN1 genes, which yields a canavanine and 5-FOA resistant strain (canR 5-FOAR). PSF3 is the first essential gene on ChrXV-L. HRI and HRII denote two regions of homology that are centered around the PAU20 and HXT11 genes, respectively.(B) PFGE analysis of ChrXV-L terminal restriction fragments following PmeI digestion of genomic DNA isolated from either the parent strain (W) or strains that have undergone a GCR event (1–9). Asterisk indicates incomplete digestion products of ChrXV.(C) Array-based comparative genome hybridization of two strains that have undergone a GCR event at ChrXV-L. The above panel is a histogram representation of log2-tranformed relative signal enrichments on Chromosome XV viewed in the University of California Santa Cruz genome browser [69]. The location of the array probes (reporters) is also indicated. Note the large loss of sequences on the left arm of ChrXV in both strains. The lower panel zooms to the ChrXV-L subtelomeric region. The breakpoint in strain (i) must be in the vicinity of the PAU20 gene (YOL161C), whereas that of strain (ii) resides in the vicinity of HXT11.
Mentions: Most of the current knowledge regarding GCR formation originates from candidate gene studies examining rearrangements at a single locus in budding yeast, the left arm of Chromosome V (ChrV-L). Although this locus has been instrumental in the deciphering of many basic mechanisms governing genome stability in eukaryotes, examination of GCR formation at other loci provides a complementary view. For example, the use of yeast artificial chromosomes to study GCRs led to the discovery that defective chromosome condensation (in a ycs4 mutant) results in GCR events [7]. In addition, studies employing a Chromosome VII disome found that defects in DNA replication and checkpoint control elevate rates of chromosome loss and rearrangements following replication fork stalling [11]. In another study, Hackett et al. employed the telomeric region of ChrXV-L to study GCR events triggered by telomerase dysfunction [12]. This latter locus is particularly useful since GCRs at ChrXV-L involve break-induced replication (BIR), a type of homologous recombination repair predicted to be a major source of genome rearrangements [2,13–15]. In contrast, GCRs formed at ChrV-L are primarily the consequence of de novo telomere addition [8]. This difference can be explained by the architecture of the telomere-proximal region on ChrXV-L, which contains two regions of homology (HRI centered on the PAU20 gene, and HRII centered on the HXT11 gene; Figure 1A) located 12 kb and 25 kb from the telomere [12]. These regions share a high degree of sequence identity with other regions in the genome [12]. As a consequence, DNA lesions formed at loci telomeric to HRI or HRII are predominantly repaired by BIR, producing nonreciprocal translocations in haploid cells. Notably, increased repair by BIR can also lead to loss of heterozygosity in diploid genomes, which may accelerate the process of tumorigenesis by inactivation of tumor suppressor genes.

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