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Yeast assays

SR Samiur Rahman
MB Mahtab Beikzadeh
ML Michael P. Latham
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C-terminal 13 × myc-tagged mre11 mutants were constructed in the integrated GAL10-HO cassette W303 S. cerevisiae strain (MATa leu2::GAL-HO-LEU2 hml ∆hmr ∆RAD5). The kanMX gene was cloned from pFA6a-kanMX vector (Addgene plasmid number 39296)65 and juxtaposed to the 3′ end of the mre11 gene for geneticin (G418 di-sulfate salt from Sigma) antibiotic selectivity. The mre11∆ strain was prepared by replacing the mre11 open reading frame with the kanMX gene. The sgs1Δ strain was prepared by replacing the sgs1 open reading frame with the trp1 gene, which was cloned from the pRS414 vector (ATCC). Verification of the integrated genes was accomplished by PCR (mre11 and sgs1) and western blot (mre11). The genotoxin sensitivity assay, NHEJ repair assay48,49, and real time PCR-based end resection assay50,51 were all performed as previously described34.

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