The influence of BRCA1 on the fidelity of NHEJ.
A, The strategy used to assay NHEJ fidelity. After digestion with I-SceI, precise ligation will produce an intact I-SceI site. B, C, After I-SceI cleavage, the NHEJ repair junctions were amplified by PCR with primers flanking the two I-SceI sites. PCR products [490 bp] were digested with I-SceI endonuclease to generate two fragments [170 bp and 320 bp]. The cells were treated with TSA [B] or BRCA1 siRNA [C]. D, E, The amount of precise rejoining at different time points was quantified by Gel Pro analysis. The relative levels of precise rejoining per I-SceI digestion of recovered pEPI-NHEJ were quantified accordingly and then represented as a histogram. F, Comparisons of the ratios of precise ligation between TSA treated cells, BRCA1 siRNA transfected cells and the control. TSA treatment induced a significant reduction of precise ligation in RGC5 cells compared to the BRCA1 siRNA treatment. All results were confirmed in three independent experiments. Error bars represent the standard deviation of the mean [n = 3]. Asterisks indicate statistically significant differences between the control and test cells [*P<0.05].