Extended Data Figure 6. Effect of Rad51 and/or Rad54 removal on DNA extension.

a. DNA synthesis from a deproteinized D-loop by Polδ in conjunction with Pif1 (8, 16, and 24 nM) was examined. Pif1 was at 24 nM in lane 6. The reaction products were analyzed in a native gel (left) or denaturing gel (right). b. After the D-loop reaction had proceeded for 2 min, Rad54, which is highly heat labile¹¹, ¹², was inactivated by incubation at 42°C for 20 min. DNA extension reaction and analysis were then performed by adding RPA, RFC, PCNA, Polδ and Pif1 (40 nM Pif1 and 8-min incubation). The reaction products were analyzed in a native gel (left) or denaturing gel (right). The inactivation of Rad54 was verified by examining the ATPase activity of Rad54, which decreased by ~95% compared to the unheated control (data not shown).