Fig. 1: SsdA deaminates cytosines only in ssDNA, without a strong sequence context dependence.

a DNA deamination activity of wildtype SsdA on 18-nt DNA substrates with different (underlined) –1 bases in the absence (ssDNA) or presence (dsDNA) of the complementary strand, examined in the EndoQ-mediated oligo cleavage assay⁴³. A 10-fold molar excess of the bottom strand was used to ensure no residual ssDNA. Note that the lower band (smaller product) for the CC substrate results from deamination at –1 C, which is in an AC context. b DNA deamination assay for SsdA and human APOBEC3B (A3B) on a 28-nt DNA substrate containing four cytosines in different sequence contexts, in the absence or presence of varying amounts of the complementary strand. The largest product results from deamination only at the most downstream TC target, whereas the smaller products are generated by deamination of more upstream cytosines in the dinucleotide motifs indicated on the right. The results show that both SsdA and A3B are ssDNA-specific. However, whereas A3B is TC-selective, SsdA can deaminate at all four sites. c DNA deamination assay for SsdA and A3B on a 36-nt DNA containing a single cytosine, in the absence or presence of varying amounts of the complementary strand, highlighting ssDNA-specific deaminase activity of SsdA and A3B. The SsdA concentration was 50 nM in (a, c), and 25 nM in (b). The A3B concentration was 0.5 μM and 1.0 μM in (b) and (c), respectively. Gels are representative of three replicates. Duplicate experiments are shown in Supplementary Fig. 6. Annealing of the substrate oligonucleotides into dsDNA in the presence of different amounts of the complementary (bottom) strand is assessed in Supplementary Fig. 4.