Nanoscale Structure and Dynamics of ABOBEC3G Complexes with Single-Stranded DNA

Abstract

The DNA cytosine deaminase APOBEC3G (A3G) is capable of blocking retrovirus replication by editing viral cDNA and impairing reverse transcription. However, the biophysical details of this host-pathogen interaction are unclear. Here we applied atomic force microscopy (AFM) and hybrid DNA substrates to investigate properties of A3G bound to single-stranded DNA (ssDNA). Hybrid DNA substrates included ssDNA with 5′ or 3′ ends attached to DNA duplexes (tail-DNA) and gap-DNA substrates, in which ssDNA is flanked by two double-stranded fragments. We found that A3G binds with similar efficiency to the 5′ and 3′ substrates, suggesting that ssDNA polarity is not an important factor. Additionally, we observed that A3G binds the single-stranded region of the gap-DNA substrates with the same efficiency as tail-DNA. These results demonstrate that single-stranded DNA ends are not needed for A3G binding. The protein stoichiometry does not depend on the ssDNA substrate type, but the ssDNA length modulates the stoichiometry of A3G in the complex. We applied single molecule high-speed AFM to directly visualize the dynamics of A3G in the complexes. We were able to visualize A3G sliding and protein association-dissociation events. During sliding, A3G translocated over a 69 nucleotide ssDNA segment in less than 1 second. Association-dissociation events were more complex, as dimeric A3G could dissociate from the template as a whole, or undergo a two-step process with monomers capable of sequential dissociation. We conclude that A3G monomers, dimers, and higher order oligomers can bind ssDNA substrates independent of strand polarity and availability of free ssDNA ends.

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

“…Although RNA-and ssDNA-dependent oligomerization of A3G has been observed, atomic force microscopy studies showed that A3G was able to bind and slide over ssDNA as a monomer (9,18,22,(42)(43)(44)(45)(46). In addition, a double mutant (F126A/W127A) that was predominantly monomeric in solution bound ssDNA and catalyzed deamination with 3=¡5= directionality similarly to wild-type A3G (17).…”

Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy

“…[9,10] It was reported that A3G nonspecifically binds to ssDNA and slides along ssDNA over 30 nm (69 nucleotides) without directional preference. [11,12] Interestingly, A3G deaminates CCC hot spots in a location-dependent fashion. [10] A 5' to 3' gradient of mutations in HIV RNA, which is transcribed from the minus strand DNA, was observed in vivo.…”

“…[9,13] This would arise from the 3' to 5' deamination gradient of minus strand DNA by A3G. Deamination by A3G has been analyzed by various methods, including gel shift assays, [10,14,15] singlemolecule fluorescence resonance energy transfer (FRET) spectroscopy, [11] and atomic force microscopy. [12,16] We previously demonstrated that real-time NMR spectroscopy can be utilized to monitor the deamination reaction and revealed that A3G CD2 deaminates the third cytidine of CCC much faster than the second one (CCC).…”

Genetic Analysis of the Localization of APOBEC3F to Human Immunodeficiency Virus Type 1 Virion Cores

“…An additional model was developed here by homology to a high-resolution structure of the A3G C-terminal domain (37,41) in order to add more amino side chain information than was present in the solved A3F crystal structure. This also supported that A3F L368 was close enough to A3F L306 for hydrophobic or van der Waals interactions.…”

“…The amino acid sequence of the C-terminal ␣-helix 6 starts at residue 358 in A3F (38,(40)(41)(42). By introducing a stop codon in A3F at either amino acid 350 (HA-A3F 350) or 360 (HA-A3F 360), truncated mutants lacking all of the ␣-helix 6 region of A3F were constructed ( Fig.…”