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Mutagenesis Advance Access originally published online on January 31, 2007
Mutagenesis 2007 22(2):147-153; doi:10.1093/mutage/gel071
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Alkaline unwinding flow cytometry assay to measure nucleotide excision repair

Bharat Thyagarajan, Kristin E. Anderson, Christopher J. Lessard1, Gregory Veltri2, David R. Jacobs, Aaron R. Folsom and Myron D. Gross1,*

Division of Epidemiology, University of Minnesota, Suite 300, West Bank Office Building, Minneapolis, MN 55454, USA 1Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA 2Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA

Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50 000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G1, S and G2/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being ~43% for cells in the G1 phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

* To whom correspondence should be addressed at Department of Laboratory Medicine and Pathology, University of Minnesota, MMC 609, 420 Delaware Street SE, Minneapolis, MN 55455, USA. Tel: +612 624 5417; Fax: +612 273 6994; Email: gross{at}epi.umn.edu

Received on April 1, 2006; revised on November 21, 2006; accepted on November 21, 2006.


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