Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy |
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| Authors: | Dr. Ayako Furukawa Dr. Kenji Sugase Dr. Ryo Morishita Prof. Takashi Nagata Prof. Tsutomu Kodaki Prof. Akifumi Takaori‐Kondo Prof. Akihide Ryo Prof. Masato Katahira |
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| Affiliation: | 1. Institute of Advanced Energy, Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611‐0011 (Japan);2. Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1‐1‐1 Wakayamadai, Shimamoto, Mishima, Osaka 618‐8503 (Japan);3. CellFree Sciences Co., Ltd., Ehime University Venture, Matsuyama, Ehime 790‐8577 (Japan);4. Department of Microbiology, Yokohama City University School of Medicine, 3‐9 Fukuura, Kanazawa‐ku, Yokohama 236‐0004 (Japan);5. Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin‐Kawaracho, Sakyo‐ku, Kyoto 606‐8507 (Japan) |
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| Abstract: | 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. |
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| Keywords: | deamination DNA enzyme kinetics quantitative analysis NMR spectroscopy |
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