Site-directed mutagenesis in a conserved motif of Epstein-Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases.
Sequence alignment of human herpesvirus DNases revealed that they share several conserved regions. One of these, the conserved motif D203...E225XK227 (D.EXK) in the sequence of Epstein-Barr virus (EBV) DNase, has a striking similarity to the catalytic sites of some other nucleases, including type II restriction endonucleases, lambda exonuclease and MutH. The predicted secondary structures of these three residues were shown to resemble the three catalytic residues of type II restriction endonucleases. Site-directed mutagenesis was carried out to replace each of the acidic residues near the motif by residues with different properties. All substitutions of D203, E225 and K227 were shown to cause significant reductions in nuclease activity. Six other acidic residues, within the conserved regions, were also replaced by Asn or Gln. Five of these six variants retained nuclease activity and mutant D195N alone lost nuclease activity. The four charged residues, D195, D203, E225 and K227, of EBV DNase were found to be important for nuclease activity. Biochemical analysis indicated that the preference for divalent cations was altered from Mg2+ to Mn2+ for mutant E225D. The DNA-binding abilities of D203E, E225D and E225Q were shown to be similar to that of wild-type. However, K227 mutants were found to have variable DNA-binding abilities: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability. Comparison of the biochemical properties of the corresponding substitutions among EBV DNase and type II restriction enzymes indicated that the D...EXK motif is most likely the putative catalytic centre of EBV DNase.
Liu
Ming-Tsan
MT
National Health Research Institutes, 3F No. 109, Section 6, Min-Chuan East Road, Taipei 114, Taiwan.
Hu
Hsien-Ping
HP
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan.
Hsu
Tsuey-Ying
TY
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan.
Chen
Jen-Yang
JY
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan.|National Health Research Institutes, 3F No. 109, Section 6, Min-Chuan East Road, Taipei 114, Taiwan.
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000201:enzymology* / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D008345:Manganese; D004247:DNA; D003851:Deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific