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

Biology

Keisuke Shindo¹  Ming Li²  Phillip J. Gross²  William L. Brown²  Elena Harjes²  Yongjian Lu²  Hiroshi Matsuo¹ 

[1] Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA;

关键词: APOBEC3G;    DNA cytosine deamination;    HIV restriction;    single-stranded DNA;    structure-guided mutagenesis;   

DOI  :  10.3390/biology1020260

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2012 by the authors; licensee MDPI, Basel, Switzerland.