Single-Stranded DNA Scanning and Deamination by APOBEC3G with Single-Molecule Resolution

Abstract

APOBEC3G (Apo3G) is a deoxycytidine deaminase that converts C → U on single-stranded (ss) DNA, favoring ataCCCaaa and disfavoring tttCCCttt motifs. Apo3G inactivates HIV-1 by attacking cDNA in T cells lacking the viral infectivity factor (Vif). Here, we observe Apo3G scanning and C → U product formation in real-time on ssDNA using single-molecule fluorescence. Apo3G displays short (<25 s) and long duration (25 s - 10 min) binding events about equally distributed. Mean displacements from the initial binding site are about 10 A, which vary depending on motif location and identity. Apo3G “long binders” retain contact with the DNA, but do not remain tethered at a single binding site. Instead, the time trajectories suggest a random, bidirectional motion, while contracting the ssDNA in a motif-dependent manner. Our data further indicate that the enzyme tends to scan more often in the vicinity of a hot motif, especially when located near the 5′-end. Using Cy5-labeled Pfu DNA polymerase, which binds U on ssDNA selectively, we show that Apo3G-catalyzed deaminations are strongly favored in the 5′-direction. The observation that random scanning generates non-random catalysis supports a model in which Apo3G binds in an asymmetric catalytically active orientation.