Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1

Bioconjugate Chemistry
Natalia A LebedevaO I Lavrik

Abstract

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

References

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Jan 26, 2010·Nucleic Acids Research·Thomas S DexheimerYves Pommier
Feb 1, 2011·FEBS Letters·Natalia A LebedevaOlga I Lavrik
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May 17, 2011·Biochemistry. Biokhimii︠a︡·M M KutuzovS N Khodyreva
Apr 24, 2012·Biochimie·Natalia A LebedevaOlga I Lavrik
Feb 26, 2013·Nucleic Acids Research·Hong-Zhang HeDik-Lung Ma
Apr 6, 2013·Chemical Communications : Chem Comm·Ka-Ho LeungDik-Lung Ma
May 23, 2013·Biosensors & Bioelectronics·Pia W JensenMagnus Stougaard

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Citations

Oct 1, 2016·Journal of Biomolecular Structure & Dynamics·Nikita A KuznetsovOlga I Lavrik
Sep 8, 2018·Journal of Enzyme Inhibition and Medicinal Chemistry·Anastasia O KomarovaOlga I Lavrik
Nov 29, 2016·Oncotarget·Selma M CuyaRobert C A M van Waardenburg
Jan 12, 2021·Frontiers in Cell and Developmental Biology·Nadezhda DyrkheevaOlga Lavrik
Oct 30, 2020·Diagnostics·Alexey S ChubarovDmitrii V Pyshnyi
Nov 21, 2017·ACS Chemical Biology·David L Wilson, Eric T Kool

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