title: The in vitro Biochemical Characterization of an HIV-1 Restriction Factor APOBEC3F: Importance of Loop 7 on Both CD1 and CD2 for DNA Binding and Deamination.
abstract: APOBEC3F (A3F) is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of proteins that can deaminate cytosine (C) to uracil (U) on nucleic acids. A3F is one of the four APOBEC members with two Zn-coordinated homologous cytosine deaminase (CD) domains, with the others being A3G, A3D, and A3B. Here we report the in vitro characterization of DNA binding and deaminase activities using purified wild-type and various mutant proteins of A3F from an Escherichia coli expression system. We show that even though CD1 is catalytically inactive and CD2 is the active deaminase domain, presence of CD1 on the N-terminus of CD2 enhances the deaminase activity by over an order of magnitude. This enhancement of CD2 catalytic activity is mainly through the increase of substrate single-stranded (ss) DNA binding by the N-terminal CD1 domain. We further show that the loop 7 of both CD1 and CD2 of A3F plays an important role for ssDNA binding for each individual domain, as well as for the deaminase activity of CD2 domain in the full-length A3F.
authors.affiliation: Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Qihan
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Genetic, Molecular and Cellular Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Genetic, Molecular and Cellular Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Wolfe
authors.affiliation: Molecular and Computational Biology Program, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089, USA; Center of Excellence in NanoBiophysics, University of Southern California, Los Angeles, CA 90089, USA; Norris Comprehensive Cancer Center; University of Southern California, Los Angeles, CA 90089, USA. Electronic address: xiaojiac@usc.edu.
authors.forename: Xiaojiang S
authors.initials: XS
authors.lastname: Chen
mesh_terms: D043525:Cytosine Deaminase / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D003641:Deamination / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015497:HIV-1 / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D043525:Cytosine Deaminase
keywords: APOBEC deaminases; APOBEC3F; Anti-HIV; DNA binding