Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA
Liudmila A. Abrosimova,
Nikita A. Kuznetsov,
Natalia A. Astafurova,
Anastasiia R. Samsonova,
Andrey S. Karpov,
Tatiana A. Perevyazova,
Tatiana S. Oretskaya,
Olga S. Fedorova,
Elena A. Kubareva
Affiliations
Liudmila A. Abrosimova
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia
Nikita A. Kuznetsov
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Lavrentiev Avenue 8, 630090 Novosibirsk, Russia
Natalia A. Astafurova
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia
Anastasiia R. Samsonova
SAS Synhelix, Genopole Campus 3, 4 Rue Pierre Fontaine, 91058 Evry-Courcouronnes, France
Andrey S. Karpov
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia
Tatiana A. Perevyazova
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya Str. 3, 142290 Puschino, Russia
Tatiana S. Oretskaya
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia
Olga S. Fedorova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Lavrentiev Avenue 8, 630090 Novosibirsk, Russia
Elena A. Kubareva
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia
Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.
