Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.
Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.
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猿猴病毒 40 大 T 抗原 ATP 结合区内调节位点的特异性突变。
DOI：
10.1128/jvi.65.9.4973-4984.1991
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发表时间：
1991
影响因子：
5.4
通讯作者：
Bradley,MK
中科院分区：
文献类型：
--
作者：
Weiner,BM;Bradley,MK
为了区分猴病毒40（SV 40）大T抗原（T）与ATP的结合与水解，根据我们的位点模型（M. K.布拉德利，T. F.史密斯河H. Lathrop，D. M.利文斯顿和T. A.韦伯斯特，Proc. Natl. Acad. Sci. USA 84：4026-4030，1987）。预测与磷酸镁接触的两个酸性残基变为丙氨酸。突变的T基因对病毒DNA合成和病毒粒子产生完全缺陷，并且对病毒DNA复制是显性缺陷。有缺陷的T基因编码一种稳定的产物（2905 T），可致癌转化小鼠细胞系。从转化细胞提取物中免疫沉淀的2905 T特异性结合SV 40来源DNA，令人惊讶的是，它具有ATP酶活性。构建了重组杆状病毒，用于生产和纯化突变蛋白，用于详细的生化分析。2905 T的ATP酶和解旋酶只有野生型的10%。2905 T对ATP的Km值与野生型T相同。ATP激活野生型T的ATP酶活性，但不激活2905 T的ATP酶活性。如通过凝胶带移测定所测试的，2905 T与SV 40来源DNA以及与单个位点I和II结合，其亲和力与野生型相似。然而，ATP没有调节突变T的DNA结合活性的网站II。因此，T中ATP结合位点的这种突变导致蛋白质与ATP之间相互作用的缺陷，这似乎是决定T与ATP酶的DNA结合活性状态的原因。
In an attempt to distinguish simian virus 40 (SV40) large T antigen (T) binding to ATP from hydrolysis, specific mutations were made in the ATP-binding site of T according to our model for the site (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Two acidic residues predicted to make contact with the magnesium phosphate were changed to alanines. The mutated T gene was completely defective for viral DNA synthesis and for virion production, and it was dominant defective for viral DNA replication. The defective T gene encoded a stable product (2905T) that oncogenically transformed mouse cell lines. 2905T, immunoprecipitated from transformed-cell extracts, bound SV40 origin DNA specifically and, surprisingly, it was active as an ATPase. A recombinant baculovirus was constructed for the production and purification of the mutant protein for detailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-type T. The Km of 2905T for ATP in ATPase assays was the same as the Km of wild-type T. ATP activated the ATPase activity of wild-type T, but not of 2905T. As tested by gel bandshift assay, 2905T bound to SV40 origin DNA and to individual sites I and II with affinities similar to that of the wild type. However, ATP did not modulate the DNA-binding activity of mutant T to site II. Therefore, this mutation in the ATP-binding site in T resulted in defects in the interaction between the protein and ATP that appeared to be responsible for the determination of the active state of T for DNA binding versus ATPase.