FIGURE 7.

Involvement of TOPOIIIα, RMI1, and RMI2 in DNA end resection. A, SDS-PAGE analysis of purified TRR complex (1.5 μg) and BLM (0.5 μg). The gel was stained with Coomassie Brilliant Blue R-250. The molecular weights of protein standards are indicated on the left. B, stimulation of BLM-DNA2-catalyzed DNA end resection by the TRR complex. Reactions contained 2 nm 3′-tailed pUC19 substrate, 8 nm DNA2, 10 nm BLM, 350 nm RPA, and varying concentrations of TRR. BLM and TRR were preincubated for 5 min on ice prior to addition to the reaction. Reaction products were analyzed as in Fig. 1C using the 112–133-nt probe. C, quantification of the product of reactions in B. Data are mean ± S.D. (n = 3). The data are normalized to the amount of product in the reaction containing only BLM and DNA2 (100%). D, RMI1 acts epistatically with BLM and DNA2 to promote DSB repair by SSA in human cells. Efficiency of SSA-mediated repair of I-SceI-induced DSB in U2OS/SA-GFP cells transfected with indicated siRNAs was measured as in Fig. 5E. E, Western blot analysis of extracts from U2OS/SA-GFP cells transfected with indicated siRNAs under the same conditions as for SA-GFP reporter assays. Blots were probed with the indicated antibodies.