Fig. 5. Brca2^L2431P mutant MEFs are proficient in RAD51 loading at low-dose camptothecin-induced DSBs.

a Schematic representation of how an unrepaired single strand break generated by Topoisomerase inhibitor (camptothecin) gets converted into a DSB during DNA replication. The undamaged sister chromatid is present near the DSB, which acts a template for HR-mediated DSB repair. b quantification of γH2AX positive nuclei in MEFs co-stained with EdU after 10 Gy IR, 30 nM camptothecin, and 1 μM camptothecin treatment for 1 h grown in 1 mM EdU media. Notably, more than 95% of DSBs seen after 30 nM camptothecin treatment is in S-phase (similar to control) as opposed to other treatments. c Representative immunofluorescence images of RAD51 foci in MEFs of indicated genotypes after 30 nM camptothecin treatment for 24 h. DSBs are marked with γH2AX, and nuclei are stained with DAPI (scale bar = 5 μm). d Quantification of RAD51-positive nuclei per γH2AX positive nuclei in MEFs after different treatments. RAD51 loading after IR is significantly reduced in LP/LP and LP/KO MEFs compared to WT (n = 3 biological replicate, two-tailed Student’s t test, error bar- SD of mean). Mutant MEFs show increased number of RAD51-positive nuclei after camptothecin and olaparib treatment (error bar- SD of mean). e Representative immunofluorescence images of RAD51 foci in MEFs of indicated genotypes after 10 nM olaparib treatment for 24 h. DSBs are marked with γH2AX, and nuclei are stained with DAPI (scale bar = 5 μm). f Quantification of chromosomal aberration observed in untreated, 30 nM camptothecin and 10 nM olaparib treated MEFs of indicated genotypes (n = 3 individual MEFs with 15 nuclei each making 45 nuclei in total, ns not significant, two-tailed Student’s t test, error bar- SD of mean).