Deaminase Activity on Single-stranded DNA (ssDNA) Occurs in Vitro when APOBEC3G Cytidine Deaminase Forms Homotetramers and Higher-order Complexes

APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2011-09, Vol.286 (35), p.30655-30661
Main Authors: McDougall, William M., Okany, Chinelo, Smith, Harold C.
Format: Article
Language:English
Subjects:
APOBEC
APOBEC-3G Deaminase
Cross-Linking Reagents - chemistry
Cross-Linking Reagents - pharmacology
Crystallization
Cytidine Deaminase - chemistry
Cytidine Deaminase - metabolism
Deaminase
Dimerization
DNA - chemistry
DNA Enzymes
DNA, Single-Stranded - metabolism
DNA-binding Protein
Dose-Response Relationship, Drug
Enzymology
Glycerol - chemistry
HIV
Host Defense
Humans
In Vitro Techniques
Protein Binding
Protein DNA-Interaction
Protein Interaction Mapping
Protein-Protein Interaction
Temperature
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:APOBEC3G (A3G) is a cytidine deaminase that catalyzes deamination of deoxycytidine (dC) on single-stranded DNA (ssDNA). The oligomeric state of A3G required to support deaminase activity remains unknown. We show under defined in vitro conditions that full-length and native A3G formed complexes with ssDNA in an A3G concentration-dependent but temperature-independent manner. Complexes assembled and maintained at 4 °C did not have significant deaminase activity, but their enzymatic function could be restored by subsequent incubation at 37 °C. This approach enabled complexes of a defined size range to be isolated and subsequently evaluated for their contribution to enzymatic activity. The composition of A3G bound to ssDNA was determined by protein-protein chemical cross-linking. A3G-ssDNA complexes of 16 S were necessary for deaminase activity and consisted of cross-linked A3G homotetramers and homodimers. At lower concentrations, A3G only formed 5.8 S homodimers on ssDNA with low deaminase activity. Monomeric A3G was not identified in 5.8 S or 16 S complexes. We propose that deaminase-dependent antiviral activity of A3G in vivo may require a critical concentration of A3G in viral particles that will promote oligomerization on ssDNA during reverse transcription.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111.269506