title: RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates.
abstract: APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181-194 in the N-terminus and aa 314-320 and 345-374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15-29, 41-52 and 83-99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA Center for RNA Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Polevoda
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA Center for RNA Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: McDougall
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Bradley N
authors.initials: BN
authors.lastname: Tun
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Cheung
authors.affiliation: OyaGen, Inc, Rochester BioVenture Center, 77 Ridgeland Road, Rochester, NY 14623, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Salter
authors.affiliation: Environmental Health Sciences Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Alan E
authors.initials: AE
authors.lastname: Friedman
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA Center for RNA Biology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA OyaGen, Inc, Rochester BioVenture Center, 77 Ridgeland Road, Rochester, NY 14623, USA Environmental Health Sciences Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA Center for AIDS Research, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA harold.smith@rochester.edu.
authors.forename: Harold C
authors.initials: HC
authors.lastname: Smith
mesh_terms: D000071480:APOBEC-3G Deaminase; D000495:Allosteric Site; D001665:Binding Sites; D001667:Binding, Competitive; D003564:Cytidine Deaminase / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D010455:Peptides; D012313:RNA; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase