Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto, Japan; Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto, Japan.
Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto, Japan.
Biochim Biophys Acta Gen Subj. 2020 Feb;1864(2):129346. doi: 10.1016/j.bbagen.2019.04.011. Epub 2019 Apr 13.
APOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation.
The real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions.
A3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect.
The concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA.
The discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.
APOBEC3F（A3F）是人类 APOBEC3（A3）家族胞嘧啶脱氨酶的成员之一，通过脱氨作用使病毒基因组互补 DNA 中的脱氧胞苷，从而发挥抗 HIV-1 作用。要全面了解 A3F 的脱氨行为，还需要进一步研究。
使用实时 NMR 方法和尿嘧啶-DNA 糖基化酶测定法，在不同浓度的 A3F-CTD 和 ssDNA 下追踪 A3F 的 C 端结构域（CTD）的活性。使用稳态荧光各向异性测量法，检测不同长度的 A3F-CTD 和 ssDNA 之间的结合。使用具有比野生型更高活性的 A3F-CTD N214H 突变体，有助于追踪反应。
发现 A3F-CTD 在较低的 ssDNA 浓度条件下（[A3F-CTD] > [ssDNA]），可有效地脱氨长 ssDNA 中的靶脱氧胞苷，而在较高的 ssDNA 浓度条件下（[A3F-CTD] < [ssDNA]），短 ssDNA 中的靶脱氧胞苷可被有效脱氨。与之前研究的 A3 家族成员 A3B 不同，该性质不受蛋白质和 ssDNA 浓度的影响。
A3F-CTD 和 ssDNA 底物的浓度会影响 A3F-CTD 的脱氨速率对 ssDNA 长度的依赖性。基于其与 ssDNA 的结合特征，可以合理地解释 A3F 的这种独特性质。
发现 A3F 脱氨酶的独特特性，加深了对其对抗 HIV-1 的作用机制的认识。我们的策略适用于研究 A3 活性的其他方面，例如与癌症发展有关的方面。
