Mazumder A, Gupta M, Pommier Y
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892.
Nucleic Acids Res. 1994 Oct 25;22(21):4441-8. doi: 10.1093/nar/22.21.4441.
We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.
我们提供的证据表明，HIV-1整合酶的3'加工活性受到保守CA二核苷酸3'端静电和/或空间干扰的显著影响。当切割性磷酸二酯键3'端的磷酸二酯键被甲基膦酸二酯键取代时，3'加工减少两个数量级。通过提高反应pH值，这种切割阻断可在一定程度上得到克服。对底物3'端进行标记显示水和甘油被阻断，但病毒DNA 3'-羟基受到刺激，它作为甲基膦酸二酯底物的亲核试剂。有趣的是，当末端核苷酸为3'-脱氧腺苷而非2'-脱氧腺苷时，使用磷酸二酯和甲基膦酸二酯底物形成了环状三核苷酸。此前已表明，酶活性位点的诱变会改变3'加工反应中亲核试剂的选择。综上所述，本研究结果表明，DNA主链的“诱变”也可改变亲核试剂的选择。
