Figure 3

APOBEC3A prefers hairpin substrates over other structures that are possible sources of ssDNA. (A) Graphical representation of ssDNA substrates tested. All substrates contained a TTCA motif and were 5′ Cy5 tagged. Structures with double-stranded regions were confirmed prior to use (Fig. S2). (B) Deaminase assay of 100 nM A3A, 20 nM A3A, or no A3A was incubated with 20 nM hairpin, replication fork, ssDNA gap, or bubble substrates containing 4 nt spans of ssDNA. Assays were performed and processed as in Fig. 1C. S denotes substrate band; P denotes the product band. (C) Quantification of 3 deaminase assay replicates was conducted as described in (B). P-values indicate significant differences in A3A activity comparing the hairpin and ssDNA gap substrates pairwise to the replication fork and bubble substrates by t-test. Deamination efficiency was displayed as percent cleavage of each substrate by 20 nM A3A. Error bars indicate standard error of the mean. (D) Deaminase assay of 100 nM A3A, 20 nM A3A, or no A3A with 20 nM of hairpin, replication fork, ssDNA gap, or bubble substrates with longer spans of ssDNA (10 nt). Assays were performed and processed as in Fig. 1C. S denotes substrate band; P denotes the product band. (E) Quantification of 3 replicate deaminase assays was conducted as in (D) and displayed as in (C). n.s. indicates no statistical difference was observed in activities among substrates. Full-length gel images for 3B and 3D are presented in Fig. S6.