A DNA sequence recognition loop on APOBEC3A controls substrate specificity.
APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Eric C
EC
Logue
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Nicolin
N
Bloch
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Erica
E
Dhuey
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Ruonan
R
Zhang
Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, New York, New York, United States of America.
Ping
P
Cao
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Cecile
C
Herate
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Lise
L
Chauveau
Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, New York, New York, United States of America; Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York, United States of America.
Stevan R
SR
Hubbard
Department of Microbiology, NYU School of Medicine, New York, New York, United States of America.
Nathaniel R
NR
Landau
D000071480:APOBEC-3G Deaminase; D000596:Amino Acids / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D003564:Cytidine Deaminase / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics*
D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000071480:APOBEC-3G Deaminase; C121399:APOBEC3A protein, human; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase