BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

Rasko, T., Der, A., Klement, E., Slaska-Kiss, K., Posfai, E., Medzihradszky, K. F., Marshak, D. R., Roberts, R. J. , Kiss, A. (June 2010) BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism. Nucleic Acids Res, 38 (20). pp. 7155-7166. ISSN 1362-4962 (Electronic) 0305-1048 (Linking)

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URL: https://www.ncbi.nlm.nih.gov/pubmed/20587501
DOI: 10.1093/nar/gkq567

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.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > Mapping and Rendering > Sequence Rendering
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes
organism description > bacteria > escherichia coli
Investigative techniques and equipment > spectroscopy > mass spectrometry
CSHL Authors:
Communities: CSHL labs
CSHL labs > Roberts lab
Depositing User: CSHL Librarian
Date: 29 June 2010
Date Deposited: 04 Oct 2011 15:17
Last Modified: 13 Mar 2018 13:53
PMCID: PMC2978348
Related URLs:
URI: http://repository.cshl.edu/id/eprint/15512

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