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. 2013 Dec 5;42(4):2380–2390. doi: 10.1093/nar/gkt1263

Figure 2.

Figure 2.

RECQ5 suppresses inhibitory effect of RAD51 on DNA DSBR by SSA. (A) Scheme of the SA-GFP reporter cassette. SSA-mediated repair of I-SceI-generated DSB results in the formation of a functional GFP allele. (B) Western blot analysis of extracts from HEK293/SA-GFP cells transfected with indicated siRNAs. The blots were probed with indicated antibodies. (C) Efficiency of SSA-mediated repair of I-SceI-induced DSB in HEK293/SA-GFP cells transfected with indicated siRNAs. Cells were transfected with appropriate siRNA (40 nM) 2 days before transfection of I-SceI-expressing plasmid. Percentage of GFP-positive cells was determined by flow cytometry 2 days after DSB induction and taken as a measure of repair efficiency. Values plotted represent relative repair efficiency calculated as a percentage of repair efficiency measured in cells transfected with control siRNA (siLuc; 100%). All data points represent an average of at least three replicates with error bars indicating standard deviation. (D) Efficiency of SSA-mediated repair of I-SceI-induced DSB in U2OS/SA-GFP cells transfected with indicated siRNAs. Experiments were performed as in (C) except that the percentage of GFP-positive cells was determined 3 days after DSB induction. (E) Western blot analysis of extracts from U2OS/SA-GFP cells transfected with pcDNA3.1/HisC-based vectors expressing wild-type RECQ5 or its mutants, K58R and F666A, respectively, as fusions with an Omni-tag. The blots were probed with indicated antibodies. (F) Effect of overexpression of the wild-type and mutant forms of RECQ5 on the efficiency of SSA-mediated repair of I-SceI-induced DSB in U2OS/SA-GFP. Cells were transfected with appropriate RECQ5 expression vector in combination with the plasmid expressing I-SceI. Percentage of GFP-positive cells was determined 3 days after plasmid transfection. Values plotted represent relative repair efficiency calculated as a percentage of repair efficiency measured in cells transfected with empty vector. All data points represent an average of at least three replicates with error bars indicating standard deviation. SSA, single-strand annealing; DSB, double-strand break; and GFP, green fluorescent protein.