Boyer P L, Ferris A L, Clark P, Whitmer J, Frank P, Tantillo C, Arnold E, Hughes S H
ABL-Basic Research Program, NCI-Frederick Cancer and Development Center, Frederick, MD 21702-1201.
J Mol Biol. 1994 Oct 28;243(3):472-83. doi: 10.1006/jmbi.1994.1673.
We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.
我们通过定点诱变分析了人类免疫缺陷病毒1型逆转录酶（HIV-1 RT）91至157位氨基酸之间的聚合酶结构域。我们使用BspMI盒构建了一系列氨基酸替换，并检测了突变型HIV-1 RT的RNA依赖性DNA聚合酶、DNA依赖性DNA聚合酶和核糖核酸酶H活性。HIV-1 RT 91至119位氨基酸之间以及151至157位氨基酸之间的区域位于掌状亚结构域内，包括聚合酶活性位点的一部分。这些区域内的许多氨基酸已被确定直接或间接参与聚合反应，因为这些残基处的氨基酸替换会降低聚合酶活性而不影响核糖核酸酶H活性。HIV-1 RT 120至150位氨基酸之间的区域位于HIV-1聚合酶的指状亚结构域内。我们认为指状亚结构域在模板定位中起作用。该区域内的许多氨基酸替换会降低或消除聚合酶和核糖核酸酶H的功能。
