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.
Highlights
The faithful replication of the genome is necessary for maintenance of genome integrity
To ensure that the gross chromosomal rearrangement (GCR) events recovered from the simultaneous loss of CAN1 and URA3 are due to break-induced replication (BIR), we analyzed GCR events in wild-type cells by pulsed-field gel electrophoresis (PFGE) using a scheme described by Hackett et al [12]
We isolated genomic DNA from parental canS 5-FOAS cells and cells that have undergone GCR events at ChrXV-L. This DNA is digested with PmeI to liberate a terminal restriction fragment, separated by pulsedfield gel electrophoresis (PFGE), and transferred onto nitrocellulose by Southern blotting to be probed with a ChrXV-L-specific fragment (NOP8; Figure 1B)
Summary
The faithful replication of the genome is necessary for maintenance of genome integrity. Disrupting processes that ensure faithful DNA replication results in chromosome breakage, hyper-recombination, or gross chromosomal rearrangements (GCRs) [1,2,3] This relationship has been highlighted in the budding yeast S. cerevisiae, where GCRs arise at high rates in cells with defects in the S-phase checkpoint [4], DNA replication licensing [5,6], DNA replication elongation [7,8,9], chromatin assembly [10], and homologous recombination (HR) repair [8]. Defects that occur during DNA replication lead to elevated levels of DNA damage, including DNA double-strand breaks (DSBs) These lesions may serve as substrates for the illegitimate repair processes resulting in GCRs. identification of genes that prevent GCRs can potentially uncover novel genome caretakers that guard cells against the accumulation of mutations. Unbiased identification of GCR suppressors could be a useful route for discovering novel genes and pathways that participate in DNA replication
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