Sequence-specific DNA recognition by the SmaI endonuclease

Abstract

SmaI endonuclease recognizes and cleaves the sequence CCC ↓ GGG. The enzyme requires magnesium for catalysis; however, equilibrium binding assays revealed that the enzyme binds specifically to DNA in the absence of magnesium. A specific association constant of 0.9 x 108 M+1 was determined for SmaI binding to a 22-base duplex oligonucleotide. Furthermore, the K(A) was a function of the length of the DNA substrate and the enzyme exhibited an affinity of 1.2 x 109 M+1 for a 195-base pair fragment and which represented a 104-fold increase in affinity over binding to nonspecific sequences. A K(m) of 17.5 nM was estimated from kinetic assays based on cleavage of the 22-base oligonucleotide and is not significantly different from the K(D) estimated from the thermodynamic analyses. Footprinting (dimethyl sulfate and missing nucleoside) analyses revealed that SmaI interacts with each of the base pairs within the recognition sequence. Ethylation interference assays suggested that the protein contacts three adjacent phosphates on each strand of the recognition sequence. Significantly, a predicted protein contact with the phosphate 3' of the scissile bond may have implications in the mechanism of catalysis by SmaI.