A sensitive biochemical assay for the detection of uracil

Cabelof, Diane C.; Nakamura, Jun; Heydari, Ahmad R.

doi:10.1002/em.20165

Cited by 8 publications

(5 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S1A and B). This assay has been previously used to quantify uracils in genomic DNA (32)(33)(34). A fluorescence scan of a nylon membrane containing splenocyte DNA after 3 days of stimulation is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Shalhout

Haddad

Sosin

et al. 2014

Molecular and Cellular Biology

View full text Add to dashboard Cite

d Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG ؊/؊ cells, uracil levels increase by 11-to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG ؊/؊ splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. When B lymphocytes are activated through antigen presentation, they acquire hypermutations in the immunoglobulin (Ig) genes, facilitating affinity maturation of antibodies. An enzyme, activation-induced deaminase (AID), initiates these events by converting cytosines in DNA to uracil (1-4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable domain (known as somatic hypermutations [SHMs]; reviewed in references 5 and 6). Generation of uracils is also the first step in the creation of strand breaks in the switch regions of Ig genes, leading to the replacement of the constant domain with other domains such as ␥, in a process called class-switch recombination (CSR; reviewed in reference 7). AID also binds near the transcription start sites of a large number of actively transcribed genes (8) and mutates a number of additional genes, including those encoding BCL-6, MYC, PAX-5, and PIM1 (9-12). The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).There is a strong connection between expression of AID and cancers in animals. Constitutive expression of AID in mice causes T cell cancers (18). Many human...

show abstract

Section: Resultsmentioning

confidence: 99%

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Shalhout

Haddad

Sosin

et al. 2014

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…DNA was isolated from yeast strains using Qiagen (http://www.qiagen.com) gravity tip columns as per the manufacturer's protocol and assayed for uracil incorporation as described in Cabelof et al [64]. Briefly, 4 μg of DNA was blocked for 2 h at 37 °C in a 2× tris/methoxyamine buffer (final concentration: 100 mM methoxyamine [Sigma-Aldrich, http://www.sigmaaldrich.com/] and 50 mM Tris-HCl, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

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

Kanellis

Gagliardi

Banáth³

et al. 2007

PLoS Genet

View full text Add to dashboard Cite

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 abstract

“…DNA was isolated from fetal tissue by the use of QIAGEN gravity tip columns per manufacturer's protocol and assayed for uracil incorporation as described in Cabelof et al 16 In brief, 4 g of DNA was blocked for 2 hours at 37°C in a 2X Tris/methoxyamine buffer (final concentration: 100 mmol/L methoxyamine; Sigma-Aldrich); 50 mmol Tris-HCl, pH 7.4). DNA was precipitated with 7.5% volume of 4 mol/L NaCl and 4 volumes of ice-cold 100% ethanol and resuspended in TE buffer, pH 7.6.…”

Section: Uracil Detectionmentioning

confidence: 99%

Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

Cabelof

Patel

Chen

et al. 2009

Blood

Self Cite

View full text Add to dashboard Cite

Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-␤-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-␤-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is com-

show abstract

A sensitive biochemical assay for the detection of uracil

Cited by 8 publications

References 14 publications

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

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

Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

Contact Info

Product

Resources

About