title: Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution.
abstract: APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C → U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3' → 5' direction. A similar polarity observed in vivo is believed responsible for initiating localized C → T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5' hot motif, a motif-dependent DNA contraction greatest for 5' hot > 3' hot > 5' cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: Gayan
authors.initials: G
authors.lastname: Senavirathne
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Jaszczur
authors.forename: Paul A
authors.initials: PA
authors.lastname: Auerbach
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Upton
authors.forename: Linda
authors.initials: L
authors.lastname: Chelico
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
authors.forename: David
authors.initials: D
authors.lastname: Rueda
mesh_terms: D000071480:APOBEC-3G Deaminase; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003641:Deamination; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005456:Fluorescent Dyes; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase