Plasmid Construct and Protein Purification

Cloning of the Mutant hTopIIIα Expression Vector
A plasmid encoding the wild-type (WT) TOP3A cDNA24 was modified by the Quickchange XL Site-Directed Mutagenesis Kit (Agilent technologies) with the following primers to recapitulate the deletion and frameshift present in subject P1: 5′-CCCTCCGTCACACGACTGTGCAGAAGGA-3′ (T3_FS_FW) and 5′-TCCTTCTGCACAGTCGTGTGACGGAGGG-3′ (T3_FS_RW).

Expression and Purification of TRR
The previously described plasmids encoding RMI1 and RMI213 and hTopoIIIα24 were co-transformed into E. coli Rosetta 2 cells, and the complex was expressed. The cells were disrupted in buffer A (50 mM Tris-HCl [pH 7.5], 0.5 M NaCl, 10% glycerol, 0.1% IGEPAL, 2 mM β-mercaptoethanol, 40 mM imidazole, 1 mM PMSF, and protease inhibitor tablet [PI, EDTA-free, Roche]) on ice before dounce homogenization and sonication. After the removal of cell debris by centrifugation, the lysate was affinity purified on a 5 mL HisTrap HP affinity column. The complex was further purified on a 5 mL HiTrap Heparin HP column in buffer B (50 mM Tris-HCl [pH 7.5], 10% glycerol, 0.1 mM EDTA, and 1 mM DTT) with a linear gradient of 200 mM to 1 M NaCl and then gel filtered on a 120 mL HiLoad 16/600 Superdex 200 column in buffer B containing 200 mM NaCl. Expression and purification of the mutant TThr812LeufsTer101RR complex were performed in a similar manner. BLM was purified as described previously.25, 26

dHJ Dissolution
The dHJ substrate construction and dissolution reactions were carried out as described previously.14