title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism.
abstract: The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.
authors.affiliation: Institute of Biochemistry, Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, 6726 Szeged, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Raskó
authors.forename: András
authors.initials: A
authors.lastname: Dér
authors.forename: Eva
authors.initials: E
authors.lastname: Klement
authors.forename: Krystyna
authors.initials: K
authors.lastname: Slaska-Kiss
authors.forename: Eszter
authors.initials: E
authors.lastname: Pósfai
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Marshak
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
authors.forename: Antal
authors.initials: A
authors.lastname: Kiss
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases