Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies
A) Schematic of the Pot1A3GCTD fusion protein design. Pot1 (pink) is fused directly to the N-terminus of A3GCTD (blue). The ssDNA contains both Pot1 and A3G binding sites: the Pot1 site in dark gray and the A3G hotspot in light gray with the linker sequence in smaller font. The resolved adenine in the -1 pocket is colored orange and the expected deaminated cytidine is blue. B) Size exclusion binding test shows that Pot1A3GCTD binds to the ssDNA substrate. Pot1A3GCTD alone is in black, the ssDNA is in gray, and the mixture of the two is in red. C) Deamination activity using a UDG-dependent cleavage assay. The Pot1A3GCTD fusion protein has the same deamination activity as that of A3GCTD. D) Schematic and structure of the Pot1A3GCTD in complex with DNA as observed in the crystal. The dA nucleotide bound to the -1 pocket is shown in orange. Two copies of the complex observed in the asymmetric unit are shown in blue (A3G), pink (Pot1), and grey/orange (DNA). The red star (schematic) and red sphere (structure) represent the zinc ion found in the catalytic site. The inset shows the 2Fo-Fc density (1σ contour level) observed for the adenine in the -1 nucleotide-binding pocket.