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 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding 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 activity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease activities. Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His(371) with Ala. The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability. In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His(371) is the active-site residue that has an essential role in PRV DNase activity.
Ho
T Y
TY
Institute of Chinese Medical Science, China Medical College, 91 Hsueh-Shih Road, Taichung 404, Taiwan.
Wu
S L
SL
Hsiang
C H
CH
Chang
T J
TJ
Hsiang
C Y
CY
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
D004279:DNA, Viral; D003851:Deoxyribonucleases