INTERACTION OF THE MBOII RESTRICTION-ENDONUCLEASE WITH DNA

The binding of the MboII restriction endonuclease (R . MboII; ENase) t o DNA containing its recognition site was investigated using a mobilit y shift assay. R . MboII forms specific, stable and immunodetectable c omplexes with its canonical target sequence. The association constant (K-a) of R . MboII was calculated to be 2.8 x 10(9)/M, and is about 10 (4)-fold higher than the K-a value for non-specific binding. Based on results obtained after sedimentation of the R . MboII-DNA complex in a glycerol gradient and measurement of the retardation of the complexes in polyacrylamide gels, we conclude that specific binding to the cano nical sequence involves a monomer of R . MboII. DNase I footprinting h as shown that the enzyme covers 16 nucleotides of DNA on the 5'-GAAGA- 3' strand.