Please use this identifier to cite or link to this item:
Title: Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes
Authors: Mashiyama, S.T.
Courtemanche, C.
Elson-Schwab, I.
Ames, B.N.
Crott, J.
Fenech, M.
Lee, B.L.
Ong, C.N. 
Keywords: Cultured human lymphocytes
DNA damage
DNA-uracil detection
Folate deficiency
Gas chromatography-mass spectrometry
Issue Date: 2004
Citation: Mashiyama, S.T., Courtemanche, C., Elson-Schwab, I., Ames, B.N., Crott, J., Fenech, M., Lee, B.L., Ong, C.N. (2004). Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes. Analytical Biochemistry 330 (1) : 58-69. ScholarBank@NUS Repository.
Abstract: Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100pg of uracil yielded a mean uracil measurement of 110.1pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12nM, and minima at 0 and 3000nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000nM folate for 10 days, P<0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3μg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans. © 2004 Elsevier Inc. All rights reserved.
Source Title: Analytical Biochemistry
ISSN: 00032697
DOI: 10.1016/j.ab.2004.03.065
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.


checked on Oct 15, 2018


checked on Oct 15, 2018

Page view(s)

checked on Oct 6, 2018

Google ScholarTM



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.