Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells.
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.
Cahová-Kucharíková
Katerina
K
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic.
Fojta
Miroslav
M
Mozga
Tomás
T
Palecek
Emil
E
D004249:DNA Damage; D004269:DNA, Bacterial / Q000032:analysis / Q000187:drug effects / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D004278:DNA, Superhelical / Q000187:drug effects / Q000528:radiation effects; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)