title: Target site cleavage by the monomeric restriction enzyme BcnI requires translocation to a random DNA sequence and a switch in enzyme orientation.
abstract: Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Sasnauskas
authors.forename: Georgij
authors.initials: G
authors.lastname: Kostiuk
authors.forename: Gintautas
authors.initials: G
authors.lastname: Tamulaitis
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D010584:Bacteriophage phi X 174 / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D021381:Protein Transport
chemical_list: D004278:DNA, Superhelical; D004279:DNA, Viral; D004247:DNA; C035801:CCSGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific