Skip to main content
. 2016 Apr 29;44(12):5743–5757. doi: 10.1093/nar/gkw326

Figure 3.

Figure 3.

MMEJ-like DSB repair in HR-deficient cells is independent of DNA-PKcs. (A) Representative sequence alignments of PCR products obtained from HPRT negative cells treated with NT control siRNA (NT), a DNA-PKcs inhibitor (NU7441), siRNA against RAD51 (siRAD51) or siRNA against RAD51 plus NU7441 (siR+NU7441), from three independent experiments (see also Supplementary Figures S1A, S5-S6). I-SceI recognition sequence and terminal microhomologies at the break sites are highlighted. (B) Average length of deletions (bp) in different genetic backgrounds. Each dot represents an independent clone. The lines represent mean and SEM, n.s., not significant, ** P < 0.01. (C) Frequency of MMEJ at mis-repaired junctions in HPRT deletion mutants isolated from cells treated with NT control siRNA (NT), NU7441, RAD51 siRNA (siRAD51) or RAD51 and NU7441 (siRAD51+NU7441). P values calculated by statistical analysis ‘‘difference between proportions’’, *P < 0.05. (D) Schematic map of the EJ2-GFP reporter (53) to assess MMEJ efficacy, where the I-SceI cut site is flanked by 8 nucleotide homologous sequences capable of bridging the I-SceI-induced DSB by MMEJ and thus restoring a functional GFP cassette. (E) MMEJ repair efficacy of EJ2-GFP reporter cells treated with NT control siRNA (NT), BRCA2 siRNA (siBRCA2), DNA-PKcs inhibitor (NU7441) or BRCA2 and DNA-PKcs co-depleted cells (siBRCA2+NU7441), indicated by the percentage of GFP-positive cells. Error bars show SEM from three independent experiments. *P < 0.05. (F) MMEJ repair efficacy of EJ2-GFP reporter cells treated with NT control siRNA (NT), BRCA1 siRNA (siBRCA1), DNA-PKcs inhibitor (NU7441) or BRCA1 siRNA and DNA-PKcs (siBRCA1+NU7441), indicated by the percentage of GFP-positive cells. Error bars show SEM from three independent experiments. *P < 0.05, ** P < 0.01.