The local dinucleotide preference of APOBEC3G can be altered from 5'-CC to 5'-TC by a single amino acid substitution.

APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5'-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5'-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5'-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5'-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5'-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained human immunodeficiency virus type 1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as human immunodeficiency virus type 1 cDNA.