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

被引:2
作者
Wan, Li [1 ,2 ]
Kamba, Keisuke [1 ]
Nagata, Takashi [1 ,2 ]
Katahira, Masato [1 ,2 ]
机构
[1] Kyoto Univ, Inst Adv Energy, Uji, Kyoto, Japan
[2] Kyoto Univ, Grad Sch Energy Sci, Uji, Kyoto, Japan
来源
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS | 2020年 / 1864卷 / 02期
关键词
Real-time NMR; APOBEC3F; Deamination; ssDNA; Activity; REAL-TIME NMR; CYTIDINE DEAMINASE; CRYSTAL-STRUCTURE; HIV-1; RESTRICTION; BINDING; FAMILY; ENZYME; MUTAGENESIS; IMPACT;
D O I
10.1016/j.bbagen.2019.04.011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: 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. Methods: 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. Results: 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. Conclusions: 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. General significance: 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.
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页数:8
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