國家衛生研究院 NHRI:Item 3990099045/2853
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    題名: Site-directed mutagpnesis in a conserved motif of Epstein-Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases
    作者: Liu, MT;Hu, HP;Hsu, TY;Chen, JY
    貢獻者: National Institute of Cancer Research
    摘要: 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 E2250 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.
    關鍵詞: Biotechnology & Applied Microbiology;Virology
    日期: 2003-03
    關聯: Journal of General Virology. 2003 Mar;84(3):677-686.
    Link to: http://dx.doi.org/10.1099/vir.0.18739-0
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0022-1317&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000181237500021
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0037373496
    顯示於類別:[陳振陽] 期刊論文

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