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. 2021 Jan 15;49(3):1436–1454. doi: 10.1093/nar/gkaa1265

Figure 4.

Figure 4.

RAD50 is required for homologous recombination. (A) Schematic showing the Quantitative qPCR assay to measure DNA resection in trypanosomes. Design of qPCR primers (black arrow) to measure ssDNA at a HindIII site upstream of the I-SceI recognition site on Chromosome 11. The mock HindIII digestion is used as a control. The primer pairs flank the HindIII site. Green arrow depicts where a PCR product will be formed and a red cross where no PCR product will be formed. Schematic created with BioRender.com (B) Schematic shows the position of the HindIII site in the RFP:PAC fusion gene (red box) and UTRs (grey box) and qPCR primers (black arrow). Histogram showing % ssDNA in the 1HR, 1HRrad50 and 1HRmre11 cell lines. I-SceI cleavage was induced with tetracycline and genomic DNA extracted at 0, 6 and 12 h for qPCR analysis of end resection. Error bars = SD n = 3 for 1HR and n = 4 for 1HRrad50 and 1HRmre11 (C–E) PCR analysis of repaired subclones showing RFP:PAC presence or absence. (C) 1HR, (D) 1HRrad50, (E) 1HRmre11, (F) percentage of repair pathway by MMEJ in the 1HR, 1HRrad50 and 1HRmre11 cell lines based on the PCR analysis of the repaired subclones.