Fig 2: MiCas9’s beneficial effects are Brex27 and RAD51 dependent.a Indel rates by spCas9, miCas9, and miCas9-mutant at the on-target site for sg-AAVS1 and the off-target site 1 for sg-EMX1. b Indel rates estimated by T7E1 assay by spCas9 and miCas9 with or without the use of RAD51 inhibitor, B02. c Co-IP results by using anti-flag (left) or anti-RAD51 (right) antibodies followed western blot for RAD51 or Cas9. d ChIP assay results with anti-RAD51 antibody at the proximal region of the sg-AAVS1 target locus. Sp: spCas9, Mi: miCas9, Mi-mut: miCas9-mutant. #Reads: Average amplicon reads per sample. Three independent experiments were performed for each condition. Data are presented as mean ± standard error of means (SEM). Unpaired t-test (two tailed) was used to compare data using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA). Source data are available in the Source Data file.

Fig 3: Bractoppin Selectively Interrupts BRCA1-Dependent Steps in DNA Repair by Homologous Recombination(A) Confocal images depicting at high magnification the recruitment of the RPA32 protein into nuclear foci after the indicated treatments. Experiments were carried out as described as in Figure 5A. Staining in the upper row is for RPA32 (green), middle row, for mCherry-BRCA1 tBRCT (red); lower row, merged red and green staining, with DNA staining (DAPI) in blue. Scale bar represents 10 µm.(B) Recruitment of RAD51 protein into nuclear foci, measured and depicted as described in (A). Scale bar represents 10 µm.(C) Percentage of cells positive for radiation-induced nuclear RPA32 foci (mean ± SD; n = 5,300, 0 Gy; 3,200, 16 Gy; 3,600, BRCA1 tBRCT; 3,400, Bractoppin; 3,500, CCBT2047) enumerated by high-content imaging (see the STAR Methods). Statistical significance was performed using an unpaired two-tailed t test. ***p = 0.001. Similar results were observed in three independent repeats.(D) Percentage of cells containing nuclear RAD51 foci enumerated and depicted as described in (B). ***p = 0.001.

Fig 4: DSB-triggered hMSH5 loading on chromatin requires hMRE11 and hRad51.(A) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. (B) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. (C) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

Fig 5: Polq is required for Pyridostatin induced DNA damage repair. a Colony forming efficiency after treatment with Pyridostatin (PDS, 1, 2, 5 µM) in WT and Polq-/- MEFs. b Colony forming efficiency after treatment with Pyridostatin (PDS, 1, 2, 5 µM) in WT MEFs transduced with Cas9 and either sgCtrl or sgPolq (targeting the polymerase domain). a, b Data shown are the mean ± SEM (n = 3). Statistical significance was assessed by two-tailed t-tests. *p < 0.05, **p < 0.01 and ***p < 0.001. c IF images for DAPI (blue), Rad51 (red) and 53BP1 (green) in WT and Polq-/- MEFs six hours after treatment with 5 µM PDS (n = 3 biologically independent experiments). d, e are quantification of large Rad51 and 53BP1 foci, as observed in (c). Shown are mean ± SEM, and representative of three independent experimental replicates. Statistical significance was assessed by Mann–Whitney tests. **p < 0.01, ***p < 0.001, and ****p < 0.0001