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.
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.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.|Department of Natural Sciences, Novosibirsk State University , 2 Pirogov Street, Novosibirsk 630090, Russia.
Natalia A
NA
Lebedeva
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.|Department of Natural Sciences, Novosibirsk State University , 2 Pirogov Street, Novosibirsk 630090, Russia.
Rashid O
RO
Anarbaev
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.
Maxim S
MS
Kupryushkin
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.|Department of Natural Sciences, Novosibirsk State University , 2 Pirogov Street, Novosibirsk 630090, Russia.
Nadejda I
NI
Rechkunova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.|Department of Natural Sciences, Novosibirsk State University , 2 Pirogov Street, Novosibirsk 630090, Russia.
Dmitrii V
DV
Pyshnyi
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.
Dmitry A
DA
Stetsenko
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences , 8 Lavrentiev Avenue, Novosibirsk 630090, Russia.|Department of Natural Sciences, Novosibirsk State University , 2 Pirogov Street, Novosibirsk 630090, Russia.
Olga I
OI
Lavrik
D001080:Apurinic Acid / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011119:Polynucleotides / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase