Cloning, Expression, and Purification of a Thermostable Nonhomodimeric Restriction Enzyme, BslI

AUTOR(ES)

Hsieh, Pei-chung

FONTE

American Society for Microbiology

RESUMO

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 bslIRα gene, and the bslIRβ gene. The α and β 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 (αβ), heterotetramers (α2β2), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (α2β2). 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 α2β2. M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the β 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 α subunit of BslI endonuclease.

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