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.

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.