Johnson Allison A, Sayer Jane M, Yagi Haruhiko, Kalena Govind P, Amin Ronak, Jerina Donald M, Pommier Yves
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, and Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.
J Biol Chem. 2004 Feb 27;279(9):7947-55. doi: 10.1074/jbc.M311263200. Epub 2003 Nov 18.
The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.
病毒蛋白HIV-1整合酶对于将病毒基因组插入人类染色体以及病毒复制是必需的。整合过程通过两个连续的整合酶介导的反应进行：3'-加工和链转移。为了研究整合酶相对于已知整合酶作用位点的DNA小沟相互作用，我们合成了含有已知绝对构型的单共价加合物的寡脱氧核苷酸，这些加合物来自苯并[a]芘7,8-二醇9,10-环氧化物在双链序列中特定位置被脱氧鸟苷的外环2-氨基基团反式开环，该双链序列对应于病毒U5 DNA的末端。由于从小沟中含有这些脱氧鸟苷加合物的双链寡核苷酸的NMR溶液结构中已知烃的取向，因此可以对小沟配体的位置与整合酶相互作用之间的关系进行详细分析。置于距离3'-加工位点两三个核苷酸的DNA小沟中的加合物抑制了3'-加工和链转移。肌苷取代表明，鸟嘌呤2-氨基基团对于在这些位置之一进行有效的3'-加工以及在另一个位置进行有效的链转移是必需的。对DNA底物两条链上整合位点的定位表明，加合物既在小沟结合位点特异性抑制链转移，又在距离加合物多达六个核苷酸的位点增强整合。这些实验证明了位置特异性小沟接触对于整合酶介导的3'-加工和链转移反应的重要性。
