Evaluation of APOBEC3B Recognition Motifs by NMR Reveals Preferred Substrates

APOBEC3B (A3B) deamination activity on ssDNA is considered a contributing factor to tumor heterogeneity and drug resistance in a number of human cancers. Despite its clinical impact, little is known about A3B ssDNA substrate preference. We have used nuclear magnetic resonance to monitor the catalytic turnover of A3B substrates in real-time. This study reports preferred nucleotide sequences for A3B substrates, including optimized 4-mer oligonucleotides, and reveals a breadth of substrate recognition that includes DNA sequences known to be mutated in drug-resistant cancer clones. Our results are consistent with available clinical and structural data and may inform the design of substrate-based A3B inhibitors.


Protein cloning, expression, and purification
The coding sequence for residues 193-382 of A3B CTD was amplified by PCR and cloned into a pET28a vector that encodes a C-terminal 6X His-tag. The A3B CTD construct 193-382 was expressed in BL21 DE3 Escherichia coli cells that were grown in LB medium + Kanamycin to an OD of 0.6 and induced overnight with 0.3 mM IPTG at 18 °C. The cells were then centrifuged and the dried cell pellet corresponding to three liters of cell culture was snap frozen in dry ice and stored at -80°C. Cell pellets were then resuspended in 5 volumes of cold lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 5% (v/v) Glycerol, 20mM Imidazole) containing 1x complete EDTA-free protease inhibitors. The cells were lysed using an Emulsiflex C3 cell homogenizer at a pressure between 10000 and 15000 psi. To ensure proper lysis was occurring, the cell suspension was passed four times through the cell homogenizer. The lysate was then clarified by centrifugation at 60000 RCF for 40 minutes at 4°C. Following centrifugation, the supernatant was loaded on a 5 mL HisTrap column previously equilibrated on lysis buffer.
Nucleic acid contamination was removed by washing the resin-bound material with lysis buffer + 1M KCl. Non-specifically bound material was removed by washing with 5 column volumes of lysis buffer plus 50 mM imidazole. Elution was performed in lysis buffer containing 250 mM imidazole.
The HisTrap eluate was subsequently concentrated to 5.0 mL and applied to a Superdex 75 HR 26/60 column equilibrated in 25 mM Hepes pH 7.5, 150 mM NaCl, 1 mM DTT. Liquid chromatography/mass spectrometry (LC/MS) was carried out on the protein as described in Newbatt et al. 1 Fractions containing pure A3B CTD were pooled, concentrated to 10 mg/mL, snap frozen in dry ice and stored at -80 °C. ssDNA ssDNA were synthesized and HPLC purified by Dharmacon, Inc (up to four nucleotides) and Eurofins Genomics (longer than four nucleotides).

Real-time NMR spectroscopy
ssDNA oligonucleotide solids were dissolved in D2O to obtain a stock solution.
The D2O stock was added to 25 mM Hepes buffer (pH 7.5 with 150 mM NaCl and 4 mM DTT). The deamination reaction was started by adding a 100 µM A3B CTD stock in the same buffer to this solution. The final composition of the reaction mixture contained 0.5 mM ssDNA oligonucleotide and 2 µM A3B CTD, a ratio of 250:1. All NMR data were recorded using a Bruker Avance 500 spectrometer equipped with a 1.7 mm TXI microprobe (Bruker biospin). Sequential 1 H-NMR spectra were acquired at 295 K every 10 minutes with the same NMR sample resident in the magnet. The initial rates of the deamination reactions were determined from the intensity of the H-5 resonance of the dC nucleotide (example spectra in Figures S4, S5 and S6), which were plotted as a function of reaction time. All data were analysed using MestReNova 12 reaction monitor module, the detailed data fitting method is demonstrated in Figure S7. At least two independent measurements were carried out (Table S1).

Fluorescence-based deamination assay
The fluorescence-based DNA cytosine deamination assay 2 was carried out (n=2) in a 384 well black proxiplate (Perkin Elmer, part number 6008289).
Recombinant human A3B CTD protein 193-382aa was diluted with assay buffer

Comment
Sequence n Initial rate ± SD (mM*h -1 ) 10-mer benchmark 5'-TTATTCATAT-3' 3 0.174 ± 0.042 Reported preferred A3B substrate 3,4 5'-TCA-3'      Figure S7. 5'-TTATTCATAT-3' deamination data analysis exemplar demonstrating data fitting by Mnova 12 reaction monitor module. 1. A series of 1 H-NMR spectra obtained at different time points from the same sample were loaded to generate the stacked spectra. 2. Two regions were selected manually to cover the NMR signal of the H-5 resonance of the dC nucleotide and the corresponding signal of the dU nucleotide.
3. The integral of the dC signal from the first spectrum (time 0) was calibrated to represent 0.5 mM of concentration and all other integrals were referenced to this value using Response Factor mode. 4. The time points were generated automatically from data acquisition time stamp. 5. The Concentrations were plotted as a function of reaction time; the data was fitted with Linear Fit Function using only data points within 30% substrate conversion. 6. The dC reduction rate was reported as the deamination rate.
First spectrum