Quantitative Analysis of Location- and Sequence-Dependent Deamination by APOBEC3G Using Real-Time NMR Spectroscopy
Quantitative Analysis of Location- and Sequence-Dependent Deamination by APOBEC3G Using Real-Time NMR Spectroscopy
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DOI：
10.1002/anie.201309940
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发表时间：
2014-02-24
影响因子：
16.6
通讯作者：
Katahira, Masato
中科院分区：
文献类型：
--
作者：
Furukawa, Ayako;Sugase, Kenji;Katahira, Masato
人类抗逆转录病毒因子 APOBEC3G (A3G) 使新合成的人类免疫缺陷病毒 1 (HIV-1) 负链脱氨基，从而消除病毒感染因子 (Vif) 缺陷的 HIV-1 毒株的感染性。([1-6]) A3G 的一个独特特性是它使位于附近的 CCC 热点脱氨基。 与不太靠近 5' 端的连接相比，更有效地连接到 5' 端。然而，这一过程的机制尚不清楚，因为它包括 A3G 与 DNA 的非特异性结合以及 A3G 沿着 DNA 链的滑动。因此，现有的方法无法利用米氏理论来分析这一过程。开发了一种新的实时核磁共振方法来明确检查非特异性结合和滑动过程，并将其应用于 A3G 脱氨基分析。因此，位置依赖性脱氨基可以通过催化速率的差异来解释，催化速率取决于 A3G 接近目标胞苷的方向。实时核磁共振实验还表明，A3G 使 CCCC 串联热点脱氨，几乎没有冗余，这表明 A3G 有效地突变了分散在 HIV-1 基因组中的许多 CCC 热点。
The human antiretroviral factor APOBEC3G (A3G) deaminates the newly synthesized minus strand of the human immunodeficiency virus 1 (HIV-1), which results in the abolition of the infectivity of virus-infectivity-factor (Vif)-deficient HIV-1 strains.([1-6]) A unique property of A3G is that it deaminates a CCC hot spot that is located close to the 5' end more effectively than one that is less close to the 5' end. However, the mechanism of this process is elusive as it includes nonspecific binding of A3G to DNA and sliding of A3G along the DNA strand. Therefore, this process cannot be analyzed by existing methods using the Michaelis-Menten theory. A new real-time NMR method has been developed to examine the nonspecific binding and the sliding processes explicitly, and it was applied to the analysis of the deamination by A3G. As a result, the location-dependent deamination can be explained by a difference in the catalytic rates that depend on the direction of the approach of A3G to the target cytidine. Real-time NMR experiments also showed that A3G deaminates CCCC tandem hotspots with little redundancy, which suggests that A3G efficiently mutates many CCC hotspots that are scattered throughout the HIV-1 genome.