Mutagenesis of the DNA binding residues in bovine pancreatic DNase1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease

Abstract

The sequence selectivity of DNasei cleavage has been investigated by site-directed mutagenesis of a chemically synthesised gene. Two key DNA binding residues have been conservatively altered (Y76F and R41K) or have had their side-chains truncated (Y76A and R41A) and the effect on the cleavage of tyr T promoter DNA has been noted. It would appear from these studies that DNasei is not sensitive to minor groove width via these DNA-contacting residues, and it is suggested that DNA helical stiffness is a controlling parameter in determining DNasei sequence selectivity.