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A General Method for Quantifying Sequence Effects on Nucleobase Oxidation in DNA

  • Yelena Margolin
  • Peter C. Dedon
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

Oxidative damage to DNA has long been associated with aging and disease, with guanine serving as the primary target for oxidation owing to its low ionization potential. Emerging evidence points to a critical role for sequence context as a determinant of the guanine ionization potential and the associated chemical reactivity of the guanine, as well as the spectrum of damage products that arise from oxidation. Recent studies also suggest that the generally accepted model of oxidation hotspots in runs of guanine bases may not hold for biologically relevant oxidants. One of the primary methods used to address these important problems of sequence context utilizes gel electrophoresis to identify the location and quantity of base damage arising in model oligonucleotides. However, this approach has limited study to those agents that produce few strand breaks arising from deoxyribose oxidation, while ionizing radiation, Fenton chemistry and other biologically relevant oxidants produce sizeable proportions of both base and sugar damage. To this end, we have developed a universal method to quantify sequence context effects on nucleobase damage without interference by strand breaks from deoxyribose oxidation.

Key words

Oxidative DNA damage guanine oxidation sequence selectivity sequence context nitrosoperoxycarbonate γ-radiation hydroxyl radical strand breaks exonuclease III 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yelena Margolin
    • 1
  • Peter C. Dedon
    • 1
  1. 1.Biological Engineering Division and Center for Environmental Health ScienceMassachusetts Institute of TechnologyCambridgeUSA

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