Back to article: Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

FIGURE 10: DSB induction and repair in fission yeast. (A) Inducible I-PpoI system. I-PpoI is under the control of an anhydrotetracycline (ahTET)-inducible promoter. The ADH1 promoter drives constitutive expression of a tetracycline repressor, TetR. TetR binds to and represses the promoter of the plant viral cauliflower mosaic virus 35S (CaMV35S) carrying three Tet operators (binding site for TetR). Addition of tetracycline inducer (yellow stars) inactivates TetR and thus activates I-PpoI expression. (B) Diagram showing the I-PpoI recognition sequence, cleavage site within rDNA and typical sequence change in cells resistant to I-PpoI cleavage (ARS, autonomously replicating sequence). (C) Analysis of DSB induction by I-PpoI and resection by Southern blot, and quantification of kinetics of resection at DSB ends and at 3.2 kb from the DSB. Diagram shows I-PpoI cleavage site at the lys1 locus. (D) Diagram of the SSA assay between two partial lambda sequences. Southern blot analysis of SSA. Lambda sequence was used as a probe.

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