Cloning, Expression, and Purification of a Thermostable Nonhomodimeric Restriction Enzyme, Bsl I

Bsl I is a thermostable type II restriction endonuclease with interrupted recognition sequence CC/NNGG (/, cleavage position). TheBsl I restriction-modification system fromBacillus species was cloned and expressed inEscherichia coli . The system is encoded by three genes: the 2,739-bpBsl I methylase gene (bslIM ), thebslIR α gene, and thebslIR β gene. The α and β subunits ofBsl I can be expressed independently inE. coli in the absence ofBsl I methylase (M.Bsl I) protection.Bsl I endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated thatBsl I forms heterodimers (αβ), heterotetramers (α2 β2 ), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramerBsl I complex (α2 β2 ). 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 theBsl I endonuclease, we propose that its active form is α2 β2 . M.Bsl I 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 inBsl I sites in the first or second cytosine are resistant toBsl I digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory toBsl I digestion. Two putative zinc fingers are found in the α subunit ofBsl I endonuclease.