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Biology 2012, 1(2), 260-276; doi:10.3390/biology1020260

A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
Present address: Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Kyoto 606-8507, Japan.
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Received: 23 May 2012 / Revised: 1 July 2012 / Accepted: 14 July 2012 / Published: 2 August 2012
(This article belongs to the Special Issue Structural and Molecular Biology of HIV)
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Abstract

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. View Full-Text
Keywords: APOBEC3G; DNA cytosine deamination; HIV restriction; single-stranded DNA; structure-guided mutagenesis APOBEC3G; DNA cytosine deamination; HIV restriction; single-stranded DNA; structure-guided mutagenesis
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Shindo, K.; Li, M.; Gross, P.J.; Brown, W.L.; Harjes, E.; Lu, Y.; Matsuo, H.; Harris, R.S. A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G. Biology 2012, 1, 260-276.

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