Rescuing DNA repair activity by rewiring H-atom transfer pathway in the radical SAM enzyme, Spore Photoproduct Lyase

Cloning, expression and purification The gene coding for the SP lyase from Geobacillus thermodenitrificans (GTNG_2348) and the C140A mutant were cloned in our previous study. [1] The C140A/S76C mutant for this study was generated by site-directed mutagenesis (by QuickChange PCR mutagenesis) using the pET-M11-C140A mutant vector as template and the following primers with the modified bases indicated in italics: forward 5ʹAAATTTGATAGCTGCAAACCGAGCGCAGA-3ʹ, reverse 5ʹ-TCTGCGCTCGGTTTGCAGCTATCAAATTT-3ʹ. The sequence of the newly generated mutant was verified by DNA sequencing. All genes possess a coding sequence for an N-terminal His6-tag allowing their purification by affinity chromatography. The pET-M11-C140A/S76C mutant SP lyase construct was transformed into Escherichia coli BL21 (DE3). Expression and purification of the wild-type enzyme, C140A and C140A/S76C mutants were performed as described previously. [1] Briefly, the expression of each protein was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The harvested cells were then disrupted by sonication and the supernatants collected after ultracentrifugation. The proteins were purified by Ni-NTA affinity chromatography (Qiagen) followed by a HiTrap Heparin column step. The purity and quantity were checked by SDS-gels and absorbance measurements at 280 nm, respectively. The iron-sulfur cluster of the purified proteins was then reconstituted as described previously. [1] The proteins were finally concentrated with a 30 000-MWCO spin concentrator (Amicon, Millipore) and stored in aliquots at -80 °C.