title: Deaminase activity on single-stranded DNA (ssDNA) occurs in vitro when APOBEC3G cytidine deaminase forms homotetramers and higher-order complexes.
abstract: APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription.
authors: Department of Biochemistry and Biophysics, Rochester, New York 14642; Center for RNA Biology, Rochester, New York 14642.
authors: William M
authors: WM
authors: McDougall
authors: Department of Biochemistry and Biophysics, Rochester, New York 14642.
authors: Chinelo
authors: C
authors: Okany
authors: Department of Biochemistry and Biophysics, Rochester, New York 14642; Center for RNA Biology, Rochester, New York 14642; Cancer Center, School of Medicine and Dentistry, the University of Rochester Medical Center, Rochester, New York 14642. Electronic address: harold.smith@rochester.edu.
authors: Harold C
authors: HC
authors: Smith
mesh_terms: D000071480:APOBEC-3G Deaminase; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D003460:Crystallization; D003564:Cytidine Deaminase / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004247:DNA; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase; D005990:Glycerol