Use of DNA Repair Enzymes in Electrochemical Detection of Damage to DNA Bases in Vitro and in Cells

Kateřina Cahová-Kuchaříková, Miroslav Fojta,* Tomáš Mozga, and Emil Paleek
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic
Anal. Chem., 2005, 77 (9), pp 2920–2927
DOI: 10.1021/ac048423x
Publication Date (Web): March 26, 2005
Copyright © 2005 American Chemical Society
*

 Corresponding author. Tel.:  +4205 41517197. Fax:  +4205 41211293. E-mail:  fojta@ibp.cz.

Abstract

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.

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History

  • Published In Issue May 01, 2005
  • Received for review October 26, 2004. Accepted March 2, 2005.

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