Harris D, Lee R, Misra H S, Pandey P K, Pandey V N
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry at New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1998 Apr 28;37(17):5903-8. doi: 10.1021/bi9728452.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a dimeric enzyme consisting of p66 and p51 subunits. The functional role of the p51 subunit remains elusive since all the catalytic functions appear to be executed through the p66 subunit. We report here that the p51 subunit, in addition to providing structural support to the p66 subunit, may be involved in facilitating the loading of the p66 subunit on to the template-primer (TP). This possibility is supported by following observations: (i) Upon binding to the TP, the p51 subunit can be dissociated by acetonitrile treatment and the template-primer-bound p66 monomer alone is capable of catalyzing DNA synthesis. (ii) Photo-cross-linking of template-primer to HIV-1 RT is abolished by dissociation of the p51 subunit prior to the TP binding but remains unaffected after the TP binding step. (iii) The p66-TP covalent complex selectively generated by UV irradiation and separated by gel electrophoresis can incorporate a single nucleotide in situ upon its renaturation in the gel. (iv) Treatment of HIV-1 RT with (tert-butyldimethylsilyl)spiroaminooxathioledioside (TSAO), an inhibitor that specifically binds to the beta7 beta8 loop of p51, destabilizes the heterodimeric enzyme, resulting in the subsequent loss of DNA binding.
1型人类免疫缺陷病毒（HIV-1）逆转录酶（RT）是一种由p66和p51亚基组成的二聚体酶。由于所有催化功能似乎都是通过p66亚基执行的，p51亚基的功能作用仍然难以捉摸。我们在此报告，p51亚基除了为p66亚基提供结构支持外，可能还参与促进p66亚基加载到模板引物（TP）上。以下观察结果支持了这种可能性：（i）与TP结合后，通过乙腈处理可使p51亚基解离，仅与模板引物结合的p66单体就能催化DNA合成。（ii）在TP结合之前p51亚基解离会消除模板引物与HIV-1 RT的光交联，但在TP结合步骤之后仍不受影响。（iii）通过紫外线照射选择性生成并通过凝胶电泳分离的p66-TP共价复合物在凝胶中复性时可原位掺入单个核苷酸。（iv）用（叔丁基二甲基甲硅烷基）螺氨基氧杂硫代二糖苷（TSAO）处理HIV-1 RT，TSAO是一种特异性结合p51的β7β8环的抑制剂，会使异二聚体酶不稳定，导致随后DNA结合能力丧失。
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