Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity

A mutant of bovine pancreatic DNase I containing two additionalresidues in a loop next to C173 has been expressed in Escherichiacoli, purified and characterized biochemically. Modelling studiessuggest that the inserted arginine and glutamate side chainsof the modified loop sequence C173-R-E-G-T-V176 could contactthe bases 3' to the cleaved bond in the major groove of a boundDNA, and that up to 10 bp could interact with the enzyme andpotentially influence its cutting rate. The loop insertion mutanthas an 800-fold lower specific activity than wild-type and showsoverall cleavage characteristics similar to bovine pancreaticDNase I. Compared with the wild-type enzyme, the mutant showsa strongly enhanced preference for cutting the inverted repeat:5'-GACTT A AAGTC-3' CTGAA T TTCAG or close variants thereof.Unexpectedly for a minor groove binding protein, the preferredcutting sites in opposite strands are staggered by 1 bp in the5' direction, causing the cleavage of a TA and a TT step, respectively.This finding demonstrates that the sequence context is relativelymore important for the cutting frequency than the nature ofthe dinucleotide step of the cleaved bond, and clearly showsthat base recognition is involved in determining the sequenceselectivity of the mutant. The importance of the sequence 5'to the cleaved bond for the cutting rate suggests that the additionalmajor groove contacts may require a distortion of the DNA associatedwith a higher energy barrier, resulting in an increased selectivityfor flexible DNA sequences and a lower overall activity of themutant enzyme.