title: Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.
abstract: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity.
  
  
  
  The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used.
  
  
  
  The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed.
  
  
  
  The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: abrludmila@gmail.com.
authors.forename: Liudmila A
authors.initials: LA
authors.lastname: Abrosimova
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: kubareva@belozersky.msu.ru.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kubareva
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: anzhelamigur@mail.ru.
authors.forename: Anzhela Yu
authors.initials: AY
authors.lastname: Migur
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: saschagawschina@gmail.com.
authors.forename: Aleksandra V
authors.initials: AV
authors.lastname: Gavshina
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: venel.ale@gmail.com.
authors.forename: Aleksandra Yu
authors.initials: AY
authors.lastname: Ryazanova
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia; Institute of Molecular Biology GmbH, Ackermannweg 4, 55128 Mainz, Germany. Electronic address: norkin_1991@mail.ru.
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Norkin
authors.affiliation: Institute of Theoretical and Experimental Biophysics of Russian Academy of Science, Institutskaya str. 3, 142290, Puschino, Moscow region, Russia. Electronic address: anatolia19@mail.ru.
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Perevyazova
authors.affiliation: Institute of Biochemistry, Justus-Liebig University, Heinrich-Buff-Ring 17, D-35392 Giessen, Germany. Electronic address: wolfgang.wende@chemie.bio.uni-giessen.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wende
authors.affiliation: Department of Nuclear Physics and Biophysics, Comenius University, Mlynska dolina F1, 842 48 Bratislava, Slovakia. Electronic address: tibor.hianik@fmph.uniba.sk.
authors.forename: Tibor
authors.initials: T
authors.lastname: Hianik
authors.affiliation: Institute of Theoretical and Experimental Biophysics of Russian Academy of Science, Institutskaya str. 3, 142290, Puschino, Moscow region, Russia. Electronic address: la_zheleznaya@mail.ru.
authors.forename: Liudmila A
authors.initials: LA
authors.lastname: Zheleznaya
authors.affiliation: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia. Electronic address: oretskaya@belozersky.msu.ru.
authors.forename: Tatiana S
authors.initials: TS
authors.lastname: Oretskaya
mesh_terms: D001407:Bacillus / Q000737:chemistry; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D004247:DNA; D003850:Deoxyribonuclease I
keywords: DNA bending; Heterodimeric restriction endonuclease; Mechanism of enzyme action; Nicking endonuclease; Thickness shear mode acoustic method