Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI

Abstract

BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene. The alpha and beta subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution. Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2). M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the alpha subunit of BslI endonuclease.

FIG. 1

Gene organization of BslI R-M system. Nine conserved motifs of M.BslI were defined according to N-4 cytosine methyltransferase (15). The amino acid residues of each motif are in parentheses. The BslIRα and bslIRβ genes are located downstream of the bslIM gene.

FIG. 2

Duplex deoxyoligonucleotides challenged with BslI endonuclease. M, 10-bp DNA marker. The arrowhead indicates the migration of uncleaved substrates. The recognition sequence of BslI is CCN₇GG. (C₁,4) and (C₂,4), first and second cytosines, respectively, methylated at the N-4 position; (C₁,5) and (C₂,5), first and second cytosines, respectively, methylated at the C-5 position. Blank cells indicate no DNA methylation.

FIG. 3

DNA binding assay. Lanes 1 and 2, DNA incubated with cell extracts; lanes 3 and 4, DNA incubated with cell extracts containing the α subunit; lanes 5 and 6, DNA incubated with cell extracts containing the β subunit; lanes 7 and 8, DNA incubated with cell extracts containing the α and β subunits mixed together in vitro; lanes 9 and 10, DNA incubated with cell extracts containing the α and β subunits coexpressed in vivo; lane 11, purified BslI restriction enzyme (25 U); lane 12, DNA substrate. Lanes 1, 3, 5, 7, and 9, cell extracts diluted by one-third; lanes 2, 4, 6, 8, and 10, cell extracts diluted by one-ninth. The arrow indicates the migration of the DNA-protein complex.

FIG. 4

DNA cleavage assay. Lane 1, 1-kb DNA marker (NEB), lanes 2 and 3, pUC19 DNA digested with purified α and β subunits, respectively; lane 4, pUC19 DNA digested with reconstituted BslI (purified α and β subunits mixed); lane 5, pUC19 DNA digested with BslI purified from coexpressed cell extract; lane 6, undigested pUC19.

FIG. 5

Native PAGE and SDS-PAGE analysis of individual α and β subunits and αβ complexes. (A) In vitro reconstitution of α and β subunits of BslI endonuclease. Lane 1, 5 μg of purified BslIα subunit; lane 2, 5 μg of purified BslIβ subunit; lane 3, 5 μg of purified BslIα subunit and 5 μg of purified BslIβ subunit mixed at 4°C overnight prior to gel electrophoresis; proteins were resolved by native 10 to 20% PAGE; lane 4, copurified BslI endonuclease (6 mg/ml) resolved in a native 10 to 20% gradient gel with longer running time. (B) Two-dimensional gel electrophoresis analysis. The entire contents of lane 4 of panel A were excised and loaded onto an SDS–10% PAGE gel in the second dimension (from left to right are resolved complexes with decreasing molecular weights). M, molecular mass standard. Arrows indicate α and β subunits.

FIG. 6

Identification of a cross-linked product of BslI complex in vitro. (A) First-dimension SDS-PAGE. DTSSP-treated BslI endonuclease was subjected to electrophoresis in the presence and absence of β-mercaptoethanol on a nonreducing SDS–10% PAGE gel. Lane M, molecular mass standards; lane 1, BslI endonuclease without DTSSP treatment; lane 2, BslI endonuclease treated with DTSSP; lane 3, BslI endonuclease treated with DTSSP followed by incubation in 5% β-mercaptoethanol for 1 h at 37°C. (B) Second-dimension SDS-PAGE. The entire contents of lane 2 of the first-dimension gel were excised and incubated in Laemmli buffer containing 5% β-mercaptoethanol at 37°C for 15 min. The sliced gel was then loaded into a reducing SDS–10% PAGE gel. Lane M, molecular mass standard. BslIα and -β subunits are indicated by arrows.