Fig. 1

Generation of a hyperactive A3G-CTD variant. a Amino acid residues of wild-type A3G-CTD and the catalytically hyper-active variant, namely CTD2, are aligned. 2K3A substitutions (L234K, C243A, F310K, C321A, and C356A) are colored green, and the additional five substitutions (P200A, N236A, P247K, Q318K, and Q322A) are colored orange. b Real-time NMR deamination assay. The product (5ʹ-AATCCdeoxy-UAAA) concentration as a function of reaction time is plotted for CTD2 (red dots) and wild-type A3G-CTD (black dots) deamination at pH 7.5 with 200 nM protein and 200 µM 5ʹ-AATCCCAAA substrate. For CTD2, the first reaction reached completion within 5 h, and the second deamination (5ʹ-AATCCdeoxy-UAAA to 5ʹ-AATCdeoxy-Udeoxy-UAAA) started, thus decreasing the concentration of the initial product. c EMSA for binding of CTD2* to the 9nt ssDNA (5′-AATCCCAAA-6-FAM). CTD2*-DNA indicates position of the CTD2*–ssDNA complex, and free DNA indicates the position of protein-free ssDNA. Fluorescent-unprobed 9nt polyA (5′-AAAAAAAAA) or fluorescent-unprobed 9nt ssDNA (5′-AATCCCAAA) were added with incremental amounts in lanes 3 (25 nM), 4 (250 nM), and 5 (2500 nM) or 7 (25 nM), 8 (250 nM), and 9 (2500 nM), respectively. Uncropped gel is shown in Supplementary Fig. 5. d Each dot represents microscale thermophoresis (MST) measurement of a mixture containing fluorescent-labeled CTD2* (50 nM) and the 9nt ssDNA 5ʹ-AATCCCAAA at various concentrations including 0.12 µM, 0.24 µM, 0.48 µM, 0.97 µM, 1.95 µM, 3.90 µM, 7.81 µM, 15.62 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1 mM, 2 mM, and 4 mM. Three independent MST experiments were performed, and bars of data points represent standard error of n = 3 measurements