T4 ENDONUCLEASE-VII - IMPORTANCE OF A HISTIDINE-ASPARTATE CLUSTER WITHIN THE ZINC-BINDING DOMAIN

The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc binding region toward the N-terminal end of the prima ry sequence. In the center of this 39-amino acid section (between resi dues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster . Closely related sequences are found in three other proteins that hav e similar zinc-binding motifs. We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single ami no acids and studying the properties of the resulting N-terminal prote in A fusions. No sequence changes within the His acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc. We found that the N-ter minal aspartate residue (Asp-40) and the two histidine residues (His-4 1 and His-43) within the cluster are essential for junction-cleavage a ctivity of the proteins. However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enz ymes. We conclude that the process of cleavage can be uncoupled from t hose of binding to and distortion of the junction. It is probable that some amino acid side chains of the His-acid cluster participate in th e phosphodiester cleavage mechanism of endonuclease VII. The essential aspartate residue might be required for coordination of catalytic met al ions.