Nucleic Acid Interaction Kinetics of APOBEC3G Investigated Using Ensemble and Single Molecule Methods

Abstract

Human APOBEC3G (A3G) is a host cell cytidine deaminase capable of restricting replication of retroviruses by deaminating ss viral DNA and also by directly inhibiting reverse transcriptase (RT)-catalyzed polymerization reactions. Only about 7 A3G molecules are packaged per HIV virion. Deamination by A3G may only happen during the short period that viral (-) strand DNA is available, necessitating rapid on/off nucleic acid binding kinetics. In contrast, in order for just a few A3G molecules to inhibit DNA polymerization by RT, they have to form a “roadblock”, requiring very slow protein dissociation from DNA. Here, we use SPR and single molecule DNA stretching to investigate the DNA/A3G interaction kinetics. Our results suggest that: (i) A3G binds ssDNA with moderate cooperativity (Hill constant ∼1.5), a binding site size of ∼15 nt, and a Kd of ∼60 nM; (ii) The on/off kinetics of A3G/ssDNA is unusually slow and multi-rate; the dominant “on” component has a bimolecular rate constant of ∼105 M−1.s−1; (iii) Dissociation of A3G from ssDNA has a fast and a slow component. The fraction of the slow component and the off times increase with longer incubation over ∼100 s. Taken together, our data are consistent with the existence of both “fast” and “slow” A3G/DNA binding modes. We hypothesize that the fast mode is a feature of protein dimers, whereas the slow mode is characteristic of multimeric A3G, with protein multimerization on ssDNA occurring over an ∼100 s time period.