Ca 2+ ‐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 Ca 2+ . Class A variants are highly dependent upon Ca 2+ , having up to 300‐fold lower activity in the presence of Mg 2+ alone compared to that in the presence of Mg 2+ and Ca 2+ , 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 Ca 2+ independent. A significant proportion of this difference in Ca 2+ ‐dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild‐type, the removal of Ca 2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26‐fold in the presence of Mg 2+ and Ca 2+ , but had no effect in the presence of Mg 2+ alone. We propose that the rate‐enhancing effect of Ca 2+ 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 Ca 2+ .