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

Támas Rasko, A. Dér, E. Klement, Krystyna Ślaska-Kiss, Eszter Pósfai, Katalin F. Medzihradszky, Daniel R. Marshak, Richard J. Roberts, A. Kiss

Research output: Contribution to journalArticle

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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 secondstrand 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.

Original languageEnglish
Pages (from-to)7155-7166
Number of pages12
JournalNucleic Acids Research
Volume38
Issue number20
DOIs
Publication statusPublished - Nov 2010

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DNA Restriction Enzymes
Organism Cloning
Escherichia coli
Methyltransferases
Genes
Plasmids
DNA Restriction-Modification Enzymes
Initiator Codon
Endonucleases
Codon
Amino Acid Sequence
Mass Spectrometry
Proteins
Hydrolysis
Clone Cells
Databases
Gene Expression
GGCC-specific type II deoxyribonucleases

ASJC Scopus subject areas

  • Genetics

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BspRI restriction endonuclease : Cloning, expression in Escherichia coli and sequential cleavage mechanism. / Rasko, Támas; Dér, A.; Klement, E.; Ślaska-Kiss, Krystyna; Pósfai, Eszter; Medzihradszky, Katalin F.; Marshak, Daniel R.; Roberts, Richard J.; Kiss, A.

In: Nucleic Acids Research, Vol. 38, No. 20, 11.2010, p. 7155-7166.

Research output: Contribution to journalArticle

Rasko, Támas ; Dér, A. ; Klement, E. ; Ślaska-Kiss, Krystyna ; Pósfai, Eszter ; Medzihradszky, Katalin F. ; Marshak, Daniel R. ; Roberts, Richard J. ; Kiss, A. / BspRI restriction endonuclease : Cloning, expression in Escherichia coli and sequential cleavage mechanism. In: Nucleic Acids Research. 2010 ; Vol. 38, No. 20. pp. 7155-7166.
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