Analysis of DNA Double-Strand Break End Resection and Single-Strand Annealing in S. pombe
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DNA double-strand break (DSB) end resection is an essential step for homologous recombination. It generates 3′ single-stranded DNA needed for the loading of the strand exchange proteins and DNA damage checkpoint proteins. To study the mechanism of end resection in fission yeast, we apply a robust, quantitative and inducible assay. Resection is followed at a single per genome DSB synchronously generated by the tet-inducible I-PpoI endonuclease. An additional assay to follow resection involves recombination between two direct repeats by single-strand annealing (SSA), since SSA requires extensive resection to expose two single-strand repeats for annealing. The kinetics of resection and SSA repair are then measured using Southern blots.
Key wordsDNA double-strand break End resection Single-strand annealing Southern blotting
This work was supported by NIH grants GM080600 and GM125650.
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