Figure 5.

Oligomerization states of A3H on ssDNA and Alu RNA. (A-F) AFM imaging was done in the presence of 5 mM MgCl₂. (A-C) The oligomeric state of A3H WT alone (WT) and bound to ssDNA (WT + ssDNA) or Alu RNA (WT + Alu RNA) demonstrated that oligomerization predominantly occurred on ssDNA. (D-F) A3H Y112A/Y113A bound to ssDNA (Y112A/Y113A + ssDNA) or Alu RNA (Y112A/Y113A + Alu RNA) demonstrated that oligomerization predominantly occurred on Alu RNA. Volume distributions of A3H were converted to stoichiometries based on analysis of dimeric A3H WT (WT). Stoichiometries were plotted against percentage of total proteins. The total proteins counted were: WT, 337; WT + ssDNA, 111; Y112A/Y113A + ssDNA, 625; WT + Alu RNA, 331; Y112A/Y113A + Alu RNA 1004. Representative AFM images of A3H WT and Y112A/Y113A are shown for (B, E) ssDNA and (C, F) Alu RNA. Images are 500 × 500 nm with a height scale of 3 nm. (G) The A3H WT or Y112A/Y113A were incubated with increasing amounts of a 118 nt ssDNA and treated with RNase A. The presence of ssDNA did not enable degradation of the bound RNA, which would occur if DNA binding caused release of the RNA by A3H. These data suggest that A3H can bind both short RNAs and ssDNA simultaneously. A representative image is shown from three independent experiments.