Ca²⁺-dependent activity of human DNase I and its hyperactive variants

Abstract

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca²⁺. Class A variants are highly dependent upon Ca²⁺, having up to 300-fold lower activity in the presence of Mg²⁺ alone compared to that in the presence of Mg²⁺ and Ca²⁺, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca²⁺ independent. A significant proportion of this difference in Ca²⁺-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca²⁺ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg²⁺ and Ca²⁺, but had no effect in the presence of Mg²⁺ alone. We propose that the rate-enhancing effect of Ca²⁺ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca²⁺.