Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase

The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 n t, capable of coding a 492-residue protein. A previous study showed that PR V DNase is an alkaline exonuclease and endonuclease, exhibiting an Escheric hia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-t erminus of PRV DNase. The deleted mutants were expressed in E. coli with th e use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-bindi ng ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3'-exonuclease activ ity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease act ivities. Further chemical modification of PRV DNase revealed that the inact ivation of DNase by diethyl pyrocarbonate, which was reversible on treatmen t with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one wel l-conserved histidine residue, site-directed mutagenesis was performed to r eplace His(371) With Ala. The mutant lost most of its nuclease activity; ho wever, it still exhibited a wild-type level of DNA-binding ability. In summ ary, these results indicate that PRV DNase contains an independent DNA-bind ing domain and that His(371) is the active-site residue that has an essenti al role in PRV DNase activity.