title: Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties.
abstract: Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A's deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A's potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions.
authors.affiliation: Section on Viral Gene Regulation, Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2780, USA, Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA and HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Mithun
authors.initials: M
authors.lastname: Mitra
authors.forename: Kamil
authors.initials: K
authors.lastname: Hercík
authors.forename: In-Ja L
authors.initials: IJ
authors.lastname: Byeon
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.forename: Shawn
authors.initials: S
authors.lastname: Hill
authors.forename: Kathyrn
authors.initials: K
authors.lastname: Hinchee-Rodriguez
authors.forename: Dustin
authors.initials: D
authors.lastname: Singer
authors.forename: Chang-Hyeock
authors.initials: CH
authors.lastname: Byeon
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Charlton
authors.forename: Gabriel
authors.initials: G
authors.lastname: Nam
authors.forename: Gisela
authors.initials: G
authors.lastname: Heidecker
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.forename: Judith G
authors.initials: JG
authors.lastname: Levin
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003641:Deamination; D029968:Escherichia coli Proteins / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
chemical_list: D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011506:Proteins; C476430:SSB protein, E coli; D012313:RNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; C121399:APOBEC3A protein, human; D003564:Cytidine Deaminase