Liu M T, Hsu T Y, Lin S F, Seow S V, Liu M Y, Chen J Y, Yang C S
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
Virology. 1998 Mar 1;242(1):6-13. doi: 10.1006/viro.1997.8974.
Epstein-Barr virus (EBV) DNase possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. To map regions of EBV DNase responsible for nuclease and DNA binding activities, a series of mutant DNase polypeptides was expressed using a bacterial system for the nuclease assay and in an in vitro transcription/translation system to assay binding activity to dsDNA or ssDNA cellulose. The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding. However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities. Substitution of Phe452 and Val458 led to inactive enzymes. In the N-terminus, deletion of residues 23-28 and residues 7-61 resulted in the loss of nuclease activity but the DNA binding activities of the deleted enzymes were intermediate and low, respectively. Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected. Based on the amino acid sequence alignment of various herpesvirus DNases, we chose four highly conserved and two less well conserved regions as controls for mutagenesis studies. These six internal deletion (ID) mutants were prepared using a recombinant PCR method. Each of the polypeptides was expressed in a bacterial system for the nuclease assay and using an in vitro transcription/translation system for the DNA binding assay. DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA. These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of an intact and highly ordered structure(s) for both activities.
爱泼斯坦-巴尔病毒（EBV）脱氧核糖核酸酶具有内切核酸酶和外切核酸酶活性，并且能以双链DNA（dsDNA）和单链DNA（ssDNA）作为底物。为了确定EBV脱氧核糖核酸酶中负责核酸酶和DNA结合活性的区域，使用细菌系统表达了一系列突变的脱氧核糖核酸酶多肽用于核酸酶分析，并在体外转录/翻译系统中分析其与dsDNA或ssDNA纤维素的结合活性。结果表明，EBV脱氧核糖核酸酶的C末端（450-460位氨基酸残基）对核酸酶活性至关重要，但对DNA结合活性而言并非必需。然而，缺失441-470位氨基酸残基会导致核酸酶活性和DNA结合活性均丧失。苯丙氨酸452和缬氨酸458的替换会产生无活性的酶。在N末端，缺失23-28位氨基酸残基和7-61位氨基酸残基会导致核酸酶活性丧失，但缺失后的酶的DNA结合活性分别为中等和较低水平。亮氨酸23突变为甘氨酸会使核酸酶活性大幅降低，但其DNA结合能力不受影响。基于各种疱疹病毒脱氧核糖核酸酶的氨基酸序列比对，我们选择了四个高度保守和两个保守性稍差的区域作为诱变研究的对照。使用重组PCR方法制备了这六个内部缺失（ID）突变体。每个多肽都在细菌系统中表达用于核酸酶分析，并在体外转录/翻译系统中进行DNA结合分析。除了缺失138-152位氨基酸残基的突变体ID2保留了中等水平的DNA结合能力外，所有六个内部缺失突变体的DNA结合和核酸酶活性均被消除。这些数据表明，由于EBV脱氧核糖核酸酶不同区域的突变导致核酸酶和/或DNA结合活性丧失，因此推测这些不同区域对于维持这两种活性的完整且高度有序的结构是必需的。
