Dissecting APOBEC3G substrate specificity by nucleoside analog interference.
The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase genes encode a set of enzymes including APOBEC1 (A1), APOBEC2 (A2), APOBEC4 (A4), and APOBEC3A-H (A3A-H). Although each possesses one or more zinc binding motifs conserved among enzymes catalyzing C-->U conversion, the functions and substrate specificities of these gene products vary considerably. For example, although two closely related enzymes, A3F and A3G, both restrict HIV-1 infection in strains deficient in virus infectivity factor (vif), A3F selectively deaminates cytosine within 5'-TTCA-3' motifs in single stranded DNA, whereas A3G targets 5'-CCCA-3' sequences. In the present study we have used nucleoside analog interference mapping to probe A3G-DNA interactions throughout the enzyme-substrate complex as well as to determine which DNA structural features determine substrate specificity. Our results indicate that multiple components of nucleosides within the consensus sequence are important for substrate recognition by A3G (with base moieties being most critical), whereas deamination interference by analog substitution outside this region is minimal. Furthermore, exocyclic groups in pyrimidines 1-2 nucleotides 5' of the target cytosine were shown to dictate substrate recognition by A3G, with chemical composition at ring positions 3 and 4 found to be more important than at ring position 5. Taken together, these results provide insights into how the enzyme selects A3G hotspot motifs for deamination as well as which approaches might be best suited for forming a stable, catalytically competent cross-linked A3G-DNA complex for future structural studies.
Rausch
Jason W
JW
Retroviral Replication Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
Chelico
Linda
L
Goodman
Myron F
MF
Le Grice
Stuart F J
SF
D000071480:APOBEC-3G Deaminase; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015658:HIV Infections / Q000201:enzymology* / Q000235:genetics; D015497:HIV-1; D006801:Humans; D009705:Nucleosides / Q000737:chemistry*; D017393:RNA Editing / Q000502:physiology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity / Q000502:physiology; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001055:Apolipoproteins B; D004277:DNA, Single-Stranded; D009705:Nucleosides; D012333:RNA, Messenger; D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase; D015032:Zinc