The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

Wen-Yen Ku, Yu-Wen Liu, Ya-Chein Hsu, Chen-Chung Liao, Po-Huang Liang, Hanna S. Yuan. and Kin-Fu Chak

¹Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 11472, Republic of China, ²Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, Republic of China, ³Institute of Biochemistry, National Yang Ming University, Taipei, Taiwan 11221, Republic of China and ⁴Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 11529, Republic of China

Abstract

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni²⁺, Mg²⁺, Co²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Cu²⁺ and Zn²⁺, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.