Abstract
Human APOBEC3G (A3G) is an antiviral factor that inactivates HIV. The C-terminal domain of A3G (A3G-CTD) deaminates cytosines into uracils within single-stranded DNA (ssDNA), which is reverse-transcribed from the viral RNA genome. The deaminase activity of A3G is highly sequence-specific; the third position (underlined) of a triplet cytosine (CCC) hotspot is converted into CCU. A3G deaminates a CCC that is located close to the 5′ end of ssDNA more effectively than ones that are less close to the 5′ end, so-called 3′ → 5′ polarity. We had developed an NMR method that can be used to analyze the deamination reaction in real-time. Using this method, we previously showed that 3′ → 5′ polarity can be explained rationally by A3G-CTD's nonspecific ssDNA-binding and sliding direction-dependent deamination activities. We then demonstrated that the phosphate backbone is important for A3G-CTD to slide on the ssDNA and to exert the 3′ → 5′ polarity, probably due to an electrostatic intermolecular interaction. In this study, we investigate the pH effects on the structure, deaminase activity, and 3′ → 5′ polarity of A3G-CTD. Firstly, A3G-CTD was shown to retain the native structure in the pH range of 4.0–10.5 by CD spectroscopy. Next, deamination assaying involving real-time NMR spectroscopy for 10-mer ssDNA containing a single CCC revealed that A3G-CTD's deaminase activity decreases as the pH increases in the range of pH 6.5–12.7. This is explained by destabilization of the complex between A3G-CTD and ssDNA due to the weakened electrostatic interaction with the increase in pH. Finally, deamination assaying for 38-mer ssDNA having two CCC hotspots connected by a long poly-adenine linker showed that A3G-CTD retains the same pH deaminase activity preference toward each CCC as that toward the CCC of the 10-mer DNA. Importantly, the 3′ → 5′ polarity turned out to increase as the pH decreases in the range of 6.5–8.0. This suggests that A3G-CTD tends to continue sliding without abortion at lower pH, while A3G-CTD tends to dissociate from ssDNA during sliding at higher pH due to the weakened electrostatic interaction.
Highlights
Human APOBEC3G (A3G), which belongs to a protein family known as apolipoprotein B mRNA-editing enzyme catalytic polypeptides, exhibits cytidine deaminase activity and converts cytosines (Cs) in single-stranded DNA into uracils (Us) (Harris and Liddament, 2004; Kitamura et al, 2011; Aydin et al, 2014)
We demonstrated that A3G-CTD possesses weak deaminase activity for 5-methyl cytosine (5mC), but none for 5-hydroxymethyl cytosine (5hmC), both of which are known to be epigenetic modifications (Kamba et al, 2015)
Real-Time NMR Monitoring of Cytosine Deamination Catalyzed by A3G-CTD
Summary
Human APOBEC3G (A3G), which belongs to a protein family known as apolipoprotein B mRNA-editing enzyme catalytic polypeptides, exhibits cytidine deaminase activity and converts cytosines (Cs) in single-stranded DNA (ssDNA) into uracils (Us) (Harris and Liddament, 2004; Kitamura et al, 2011; Aydin et al, 2014). A3G-CTD is the domain responsible for C to U deamination within ssDNA, and thereby for antiviral capability (Navarro et al, 2005; Browne et al, 2009; Kobayashi et al, 2014). Both A3G-NTD and A3G-CTD possess Zn2+coordinating motif, His-X-Glu-X27−28-Pro-Cys-X2-Cys, and this motif is required for A3G-CTD to exert the catalytic activity (Harris and Liddament, 2004). How A3G-CTD interacts with ssDNA is still not well understood
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