An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA, which is affected by the concentrations of APOBEC3F and single-stranded DNA
An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA, which is affected by the concentrations of APOBEC3F and single-stranded DNA
复制标题
深入了解人 APOBEC3F 脱氨基速率对单链 DNA 长度的依赖性，该依赖性受 APOBEC3F 和单链 DNA 浓度的影响
DOI：
10.1016/j.bbagen.2019.04.011
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Katahira Masato
中科院分区：
文献类型：
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作者：
Wan Li;Kamba Keisuke;Nagata Takashi;Katahira Masato
研究背景APOBEC 3F（A3 F）是人APOBEC 3（A3）胞苷脱氨酶家族的一员，通过对病毒基因组互补DNA中的脱氧胞苷进行脱氨而发挥抗HIV-1因子的作用。方法采用实时核磁共振（real-time NMR）和尿嘧啶-DNA糖基化酶（uracil-DNA glycosylase，UCA）测定方法，研究了不同浓度的A3 F-CTD和ssDNA对A3 F C-末端结构域（CTD）活性的影响。稳态荧光各向异性测量用于检查A3 F-CTD与不同长度的ssDNA之间的结合。使用的A3 F-CTD N214 H突变体，具有更高的活性比野生型，促进了跟踪的reactions. ResultsA 3F-CTD被发现有效地脱氨的目标脱氧胞苷在较低的ssDNA浓度条件下（[A3 F-CTD][ssDNA]），而目标脱氧胞苷在短的ssDNA脱氨有效地在较高的ssDNA浓度条件下（[A3 F-CTD][ssDNA]）。这个属性是完全不同的，从以前研究的A3家族成员，A3 B;的蛋白质和单链DNA的浓度没有effect.ConclusionsThe浓度的A3 F-CTD和单链DNA底物影响的单链DNA长度依赖的脱氨率的A3 F-CTD。A_3F的这种独特性质是根据其与ssDNA的结合特性合理解释的。一般意义A_3F关于脱氨速率的独特性质的发现加深了对其抗HIV-1作用的理解。我们的策略适用于研究A3活性的其他方面，例如参与癌症发展的那些方面。
BackgroundAPOBEC3F (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.MethodsThe 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.ResultsA3F-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.ConclusionsThe 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.General significanceThe 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.