| Popis: |
Human APOBEC3G (A3G) is a host cell cytidine deaminase capable of restricting replication of retroviruses by deaminating single-stranded (ss) viral DNA, and also by directly inhibiting RT-catalyzed polymerization reactions. We have studied the kinetics of A3G interaction with model 10-, 20-, 40- and 60-nt ssDNAs as well as with long polymeric DNA by three independent approaches: (i) surface plasmon resonance (SPR) analysis, (ii) fluorescence polarization spectroscopy (FP), and (iii) single-molecule (SM) DNA stretching. Our results suggest the following: (1) Binding kinetics of A3G protein to all ssDNA oligonucleotides are multi-step with the faster, weaker binding gradually converting to more stable binding with much slower on/off rates; (2) The typical A3G-ssDNA on/off times measured in our experiments range between ∼10 and 10,000 sec, and the corresponding Kd values range between 0.1 and 10 μM; (3) Higher concentrations of A3G, longer A3G/ssDNA incubation times, as well as ssDNA length were found to correlate with slower ssDNA on/off kinetics, suggesting that A3G/A3G interactions in the bound state are responsible for slow protein kinetics.Taken together, these observations suggest that multimerization of A3G on ssDNA leads to a gradual decrease in its on/off kinetics, which in turn enhances this protein's ability to stall reverse transcription via formation of a “roadblock” for reverse transcriptase. |
