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首页> 外文期刊>Journal of Virology >Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.
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Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

机译：影响腺病毒DNA复制的热Abile DNA结合蛋白的功能表征。

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The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.
机译：先前已经显示人腺病毒型2（AD2）突变体AD2TS111含有两种导致复杂表型的突变。 AD2TS111含有在早期区域1B（E1B）19,000分子量（19K）编码区域中的单个基础变化，其产生Cyt Deg表型和另一种缺陷，其映射到导致的E2A 72K DNA结合蛋白（DBP）编码区一种温度敏感的DNA复制表型。在这里，我们报告说，AD2TS111 DBP中的缺陷是由于单一的G - -T转换，导致氨基酸在氨基酸的甘氨酸替代缬氨酸。隔离，恢复了温度无关的回复剂Ad2TS111R10。在氨基酸的甘氨酸280中还保留了由19K突变引起的Cyt和Deg表型。我们通过构建重组病毒，Ad2TS111A，含有野生型AD2 E1B 19K基因和72K基因中的vly ---- Val突变来物理分离了AD2TS111的两个突变。 AD2TS111A是Cyt + Deg +，但在非智能温度下的DNA复制仍然有缺陷。 AD2TS111 DBP突变仅位于涉及Ad2 + Nd1Ts23中的突变位点的两个氨基酸，先前测序的DBP突变体。纯化Ad2 + Nd1TS23 dBP的生化研究表明，该蛋白质缩小伸长率，但在不含纯化的Ad聚合酶，末端蛋白前体和核因子I. AD2 + ND1TS23 DBP的无细胞复制系统中，该蛋白质对伸长率无抗体引发对于单链DNA而不是AD2 DBP，如AD2 DBP，如来自变性DNA纤维素柱的纯化DBPS所示。这种对DNA的结合可能是由于蛋白质在对DNA结合的局部临界的局部构象变化而不是蛋白质结构的全局变化，因为AD2 + ND1TS23和AD2 DBP都显示出通过蛋白酶散热素的相同的切割模式在各种温度下。

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