Fluorescent probes for the analysis of DNA strand scission in base excision repair.

Nucleic Acids Research
Naoyuki MatsumotoShigenori Iwai

Abstract

We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5'- and 3'-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using (32)P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect th...Continue Reading

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Citations

Feb 5, 2014·Analytica Chimica Acta·Guillaume GinesDidier Gasparutto
Dec 19, 2013·PloS One·Aaron C JacobsR Stephen Lloyd
Jul 6, 2014·Scientific Reports·Tatsuya TogaShigenori Iwai
Dec 2, 2014·Chemical Communications : Chem Comm·Jing TaoYu Xiang
Jun 19, 2012·Stem Cell Research·Elisia D TichyPeter J Stambrook
Aug 7, 2012·Molecular Cell·Justin L SparksPeter M Burgers
May 29, 2012·Chembiochem : a European Journal of Chemical Biology·Kazumitsu OnizukaPeter A Beal
Jun 19, 2013·Current Protocols in Nucleic Acid Chemistry·Andrei P Guzaev
Aug 17, 2018·Scientific Reports·Shunsuke ItoShigenori Iwai
Nov 21, 2017·ACS Chemical Biology·David L Wilson, Eric T Kool

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Methods Mentioned

BETA
glycosylases
electrophoresis
fluorescence microscopy
antisense oligonucleotides
transfection

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