APOBEC3G (Apo3G) is a deoxycytidine deaminase that converts C->U on single stranded (ss) DNA. In T cells, Apo3G deactivates viral infectivity factor deficient (?Vif) HIV-1 strains by introducing cytosine deaminations on cDNA. Apo3G deaminations in vitro exhibit a pronounced 5'->3' bias, which is also attributed to the local mutational gradients observed in (?Vif) HIV-1 genome in vivo. Here, we investigate the ssDNA scanning and deamination mechanism of Apo3G with single-molecule resolution using F�rster resonance energy transfer (smFRET). Our data show that Apo3G scans on ssDNA bi-directionally with average excursion lengths of ~10 � and ~1 s-1 scanning rates. Apo3G quasi-localization is observed on ataCCCaaa motifs located near the 5' end, even though the enzyme spends about the same amount of time (~100 s) on other ssDNAs containing different motifs. Motif-dependent ssDNA bending is also observed, where the bending is maximal for ataCCCaaa located near the 5' end.  Interestingly, both the Apo3G scanning and Apo3G-induced ssDNA bending is reduced with lowered ionic strength, indicating that Apo3G motion on ssDNA is facilitated by salt. Consistent with the previous reports, single-molecule deamination assay demonstrates sequence- and location-dependent deamination, indicating the fluorophore-labeled protein is active on surface immobilized DNA. Although scanning is random, asymmetric catalytic orientation may be the reason for Apo3G directional deamination.

Single-stranded DNA scanning and deamination by APOBEC3G with single-molecule resolution

243rd National Meeting (2012)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.