Quantifying position-dependent deamination by APOBEC3C using real-time NMR spectroscopy

APOBEC3C (A3C) is a DNA deoxycytidine deaminase that restricts lentiviruses by converting deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). Among APOBEC3 enzymes, positional dependence has been characterized for APOBEC3G (A3G). A3G more efficiently deaminates 5'-proximal than 3'-proximal target dC residues. We previously showed that this preference can be explained by a model in which deamination is more efficient when A3G approaches a target dC in the 3'-to-5' direction than in the 5'-to-3' direction. The effect of target position on A3C-mediated deamination remains unknown. We examined this dependence using a uracil-DNA glycosylase-coupled cleavage assay and real-time nuclear magnetic resonance (NMR) spectroscopy. Analysis of 49-nt ssDNA substrates containing the same target sequence at different positions showed that the fraction of the deaminated products generally increased as the target dC was placed closer to the 3' end. We then analyzed a 26-nt ssDNA containing a 5'-proximal target at the sixth position, C6, and a 3'-proximal target at the twenty-second position, C22. Real-time NMR monitoring of the H5-H6 total correlation spectroscopy cross-peaks showed that the apparent first-order deamination rate constant for C22 was 2.2-fold greater than that for C6. Thus, A3C deaminates the 3'-proximal target more rapidly than the 5'-proximal target. This pattern is opposite to that for A3G. Structural differences in loop 7 may contribute to the opposite positional preferences of A3C and A3G. A higher catalytic rate following 5'-to-3' target approach and/or a higher sliding rate in the 5'-to-3' direction along ssDNA could underlie the positional preference of A3C.