Figure 4

APOBEC3A activity is reduced more severely on linear substrates than on hairpins in the presence of whole cell lysate or RPA. (A) Deaminase assay of 20 nM A3A or no A3A incubated with 20 nM hairpin, linear, or gap-fill substrates with or without whole cell lysate. Substrates were preincubated with 40 µg of SKBR3 whole cell lysates or buffer for 1 h at 37 °C, then A3A was added to 20 nM final concentration, and incubated for an additional 30 min at 37 °C prior to processing as in Fig. 1C. S denotes the substrate band; P denotes the product band. The results are representative of three independent experiments. (B) Coomassie stained SDS-PAGE gel of purified human RPA. (C) Electrophoretic mobility shift assay of 0, 25, 50, and 100 nM RPA incubated with 50 nM hairpin or linear ssDNA substrate. Three replicate experiments were quantified to determine the percent of each substrate bound. Dots and error bars indicate mean values and standard deviation. (D) 4 nM A3A was incubated with 50 nM hairpin or linear ssDNA substrate at 37 °C for 5 or 15 min, respectively, in the presence or absence of 100 nM RPA. The percent substrate cleavage in the presence and absence of 100 nM RPA as well as the fraction of A3A deaminase activity remaining after RPA addition for the hairpin (black dots) and linear substrates (red dots) was quantified from 4 experimental replicates. Horizonal bars indicate mean values. P-values were determined by t-test. Full-length gel images for 4A and 4D are presented in Fig. S7.