Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy

Furukawa, Ayako; Sugase, Kenji; Morishita, Ryo; Nagata, Takashi; Kodaki, Tsutomu; Takaori‐Kondo, Akifumi; Ryo, Akihide; Katahira, Masato

doi:10.1002/anie.201309940

Cited by 15 publications

(26 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the former study, we used an ssDNA substrate containing two CCC hotspots, connected by a long linker region, to monitor the deamination reaction, and revealed that A3G CD2 can deaminate the two hotspots with 3′→5′ polarity [ 28 ]. It was also revealed that the 3′→5′ polarity is caused by sliding, because the polarity disappeared when sliding was inhibited through the formation of a DNA duplex at a linker region [ 28 ]. Here, we substituted the linker region of an ssDNA substrate with a poly-ribonucleotide or poly-abasic deoxyribonucleotide and then subjected it to real-time monitoring.…”

Section: Resultsmentioning

confidence: 99%

“…Water signal suppression was achieved by 3-9-19 watergate and echo-antiecho pulse schemes in each 2D TOCSY and 2D 1 H– 13 C HSQC measurement. Resonance assignments of the oligonucleotides were carried out using 2D TOCSY and 2D 1 H– 13 C HSQC spectra [ 11 , 28 ]. Spectra were processed with NMRPipe [ 39 ], and analyzed using SPARKY [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

“…In the subsequent study, we monitored the deamination reactions of ssDNA containing two CCC hotspots and revealed that A3G CD2 can deaminate the two hotspots with 3′→5′ polarity. Furthermore, by construction of a kinetic model, in which nonspecific protein:ssDNA binding and sliding processes are incorporated, we quantitatively analyzed the 3′→5′ polarity of deamination by A3G CD2 [ 28 ]. It was revealed that the 3′→5′ polarity of A3G can be rationally explained by introducing the sliding direction-dependent catalytic rate.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

2015

Self Cite

View full text Add to dashboard Cite

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…Importantly, A3G exhibits a deamination bias, so-called 3′ → 5′ polarity. In vitro , A3G deaminates a CCC hotspot that is located close to the 5′ end more effectively than ones that are less close to the 5′ end (Chelico et al, 2006 ; Holden et al, 2008 ; Furukawa et al, 2014 ) (Figure 1A ). This phenomena is called 3′ → 5′ polarity (Chelico et al, 2006 ), because the efficiency of deamination increases in the 3′ → 5′ direction.…”

Section: Introductionmentioning

confidence: 99%

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

2016

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Residual dipolar couplings report on submicro-to millisecond dynamics [9]. Much slower dynamics on the timescale of seconds to hours can be studied by CLEANEX-PM [10,11], hydrogen-exchange [12], and real-time NMR experiments [13,14].…”

Section: Introductionmentioning

confidence: 99%

Resolving biomolecular motion and interactions by R2 and R1ρ relaxation dispersion NMR

Walinda

Morimoto

Sugase

2018

Methods

Self Cite

View full text Add to dashboard Cite

Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R and R, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.

show abstract

Quantitative Analysis of Location‐ and Sequence‐Dependent Deamination by APOBEC3G Using Real‐Time NMR Spectroscopy

Cited by 15 publications

References 22 publications

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

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

Resolving biomolecular motion and interactions by R2 and R1ρ relaxation dispersion NMR

Contact Info

Product

Resources

About