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.
Bioconjug Chem
; 26(10): 2046-53, 2015 Oct 21.
Article
em En
| MEDLINE
| ID: mdl-26335988
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
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Ácido Apurínico
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Polinucleotídeos
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Bioensaio
/
Diester Fosfórico Hidrolases
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Bioconjug Chem
Ano de publicação:
2015
Tipo de documento:
Article