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.

Figure 1.

Schematic map of the B. sphaericus DNA region carrying the genes of the BspRI R-M system. Restriction sites used in plasmid constructions are shown. (A), pES1 Plasmid. Thin line, pBR322 vector; heavy line, B. sphaericus DNA; R, bspRIR gene; M, bspRIM gene. (B) Open arrowheads show the positions of the oligonucleotide primers used in PCR reactions. (C) Beginning of the ORF encoding R.BspRI in pBAD-Bsp3. The ATG start codon of the pBAD24 vector is shown in bold. Square brackets indicate remnants of restriction sites used in construction of pBAD-Bsp3.

Figure 2.

BspRI endonuclease sequence coverage by mass spectrometry. Upper panel, MALDI–TOF mass spectrum of BspRI unfractionated tryptic digest. Numbers in brackets indicate sequence positions of the peptides. Lower panel, amino acid sequence of BspRI restriction endonuclease. Sequences identified from MS/MS data (italic) (Table 1) or from mass only are underlined.

Figure 3.

Alignment of the predicted active site motifs of BspRI and the putative REases (Table 2) sharing the highest amino acid sequence similarity with BspRI. Numbers at the beginning of the sequences designate the position of the first amino acid shown. The numbers of residues between the shown blocks of sequences are given in parentheses. Predicted α-helices and β-strands (41), are indicated at the top. The convention of numbering, with Roman numerals, the secondary structural elements of the PD-(D/E)XK conserved fold is adopted from (48). Conserved residues of the putative PD-(D/E)XK nuclease fold (47,48) are highlighted with colored background: red, acidic; grey, lysine; yellow, uncharged.

Figure 4.

Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. (A) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. (B) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. (C) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.