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Mechanisms of nucleoside analog antiviral activity and resistance during human immunodeficiency virus reverse transcription.人类免疫缺陷病毒逆转录过程中核苷类似物抗病毒活性及耐药性的机制
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Photoaffinity labeling of HIV reverse transcriptase: utilization of 2',3'-dideoxyuridylate analog bearing aryl(trifluoromethyl)diazirine moiety as a chain terminator.HIV逆转录酶的光亲和标记：利用带有芳基（三氟甲基）二氮杂环丙烷部分的2'，3'-二脱氧尿苷酸类似物作为链终止剂。
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Structure-function analysis of the mammalian DNA polymerase beta active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer.哺乳动物DNA聚合酶β活性位点的结构-功能分析：天冬氨酸256、精氨酸254和精氨酸258在核苷酸转移中的作用
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2.3 A crystal structure of the catalytic domain of DNA polymerase beta.2.3 DNA聚合酶β催化结构域的晶体结构。
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Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.利用三磷酸脱氧胸苷（dTTP）作为光亲和标记物鉴定HIV-1逆转录酶的核苷酸结合位点。
Biochemistry. 1993 Aug 3;32(30):7630-4. doi: 10.1021/bi00081a005.
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Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases.一种新型光交联CTP类似物的合成、表征及其在光亲和标记大肠杆菌和T7 RNA聚合酶中的应用。
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一种带有芳基（三氟甲基）二氮杂环丙烷部分的光不稳定2'，3'-二脱氧尿苷酸类似物：HIV-1逆转录酶的光亲和标记

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

作者信息

Yamaguchi T, Saneyoshi M

机构信息

Department of Biological Sciences, Teikyo University of Science and Technology, Yamanashi, Japan.

出版信息

Nucleic Acids Res. 1996 Sep 1;24(17):3364-9. doi: 10.1093/nar/24.17.3364.

DOI:10.1093/nar/24.17.3364
PMID:8811091
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC146106/
Abstract

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

摘要

为了开发一种用于DNA聚合酶（包括逆转录病毒逆转录酶（RT））的光亲和标记试剂，我们使用2',3'-二脱氧-E-5-[4-(3-三氟甲基-3H-重氮丙啶-3-基)苯乙烯基]尿苷三磷酸（TDSddUTP）作为底物dTTP类似物。使用放射性标记试剂（[γ-32P]TDSddUTP）和聚（A）·寡聚（dT）作为模板/引物，对人类免疫缺陷病毒1型（HIV-1）RT进行光亲和标记实验，结果因Mg2+浓度而异。在0.025 mM Mg2+存在下，光亲和标记显示，当用近紫外光（365 nm）照射时，TDSddUTP选择性地结合到p66/p51异二聚体酶蛋白66 kDa亚基中的dTTP结合位点。在4 mM Mg2+或0.05 mM Mn2+存在下，由于RT活性，TDSddUTP被掺入引物链的3'-末端，并且所得的光不稳定引物在光照射时与HIV-1 RT的66 kDa亚基结合。这些结果表明，TDSddUTP可能是研究包括HIV-1 RT在内的对该化合物具有亲和力的DNA聚合酶底物结合位点的有用工具。