Generation and Verification of RAD-resistant HSP90 Variants

The generation of the RAD-resistant HSP90 variant (HSP90rr) was described earlier^{27 (link)}. Plasmid pUC:HSP90rr served as template for site-directed mutagenesis. Primers (Supplementary data, Table S1) were phosphorylated using ATP and polynucleotide kinase (PNK, New England Biolabs)⁹⁰ and then used to prime a PCR amplification of pUC:HSP90rr using the iProof-PCR kit for GC-rich DNA (#172–5320) from Bio-Rad Laboratories (München, Germany). Following amplification (30 cycles), the linear PCR product was subjected to ligation (3 h, RT) to form circular plasmids. These were then used to transform competent E. coli DH5-α cells (#18265–017, Invitrogen, Karlsruhe, Germany). After amplification and purification⁹⁰ via caesium-chloride density gradient ultracentrifugation (50% w/v CsCl, 6 h, 90,000 rpm, 20 °C, rotor NVT90, Beckman, Krefeld, Germany), the pUC:HSP90rr-derived mutants were verified by DNA sequencing (LGC Genomics, Berlin, Germany).
The derivative of the plasmid pJC45^{91 (link)}, pJC45:HSP90, was also used for site-directed mutagenesis. The same set of primers (Table S1) was used. PCR, ligation, transformation of E. coli and CsCl purification of plasmids were performed the same as for the site-directed mutagenesis of pUC:HSP90rr.