Characterization of the deamination coupled with sliding along DNA of anti-HIV factor APOBEC3G on the basis of the pH-dependence of deamination revealed by real-time NMR monitoring

• Main Authors: Keisuke eKamba, Takashi eNagata, Masato eKatahira
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2016-04-01
• Series: Frontiers in Microbiology
• Subjects: HIV-1; enzyme; Monitoring; NMR; POBEC3G
• Online Access: http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00587/full

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.