A Screen for Suppressors of Gross Chromosomal Rearrangements Identifies a Conserved Role for PLP in Preventing DNA Lesions

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
PLoS Genetics (Impact Factor: 7.53). 09/2007; 3(8):e134. DOI: 10.1371/journal.pgen.0030134
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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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    • "LOH measures nonconservative rearrangements within or between homologous chromosomes that lead to hemi- or homozygosity of the met15Δ locus starting from a met15Δ/MET15 heterozygous diploid (Andersen et al. 2008). The GCR assay measures loss of a distal portion of a chromosome arm, typically by terminal deletion and de novo telomere addition (Smith et al. 2004; Kanellis et al. 2007). Each assay reports on loss of a genetic marker that can originate from several possible mechanisms. "
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    • "Yeast Saccharomyces cerevisiae is a model organism often used in genome stability studies. For technical reasons, including greater simplicity of molecular genetics manipulations, haploid cells were employed in the vast majority of those studies, including those employing various whole-genomic approaches [7]–[10]. However, S. cerevisiae cells can be cultivated and studied as both haploids and diploids; it has been shown that there is a two orders of magnitude difference in the frequencies of spontaneous DNA changes at CAN1 marker between a haploid genome and diploid CAN1/can1Δ heterozygous genome [11]. "
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    PLoS ONE 06/2011; 6(6):e21124. DOI:10.1371/journal.pone.0021124 · 3.23 Impact Factor
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    • "While microorganisms and plants have the ability to make the compound de novo, it is an essential nutrient in the diet of humans, being derived primarily from plant sources. Indeed, deficiency of the vitamin in humans and its association with various medical ailments has been well documented (Grillo et al., 2001; Stitt et al., 2002; Komatsu et al., 2003; Metz et al., 2003; Kanellis et al., 2007). All organisms have the ability to interconvert the different vitamer forms through a so-called salvage pathway (Figure 1). "
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    ABSTRACT: Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre-reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.
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