Dh10bac cells

pFastBac vectors with expression cassettes were transposed into the baculoviral genome using chemically competent DH10Bac cells (Thermo Fisher Scientific) according to the manufacturer’s protocol. Preparation of the baculoviral genome, transfection/P0 virus generation and P1 virus amplification were performed as described in the Bac-to-Bac manual (Thermo Fisher Scientific), with the exception of using Cellfectin® II transfection reagent and Sf-900 III serum-free medium (Thermo Fisher Scientific).

MRE11–Flag/RAD50–6×His/NBS1–Flag was prepared by co-expression in Sf9 cells. In brief, the pFastBac1 plasmids containing genes for recombinant human MRE11–Flag, RAD50–6×HIS and NBS1–Flag^{40 (link),41 (link)} were transformed into DH10Bac cells (Thermo Fisher Scientific, 10361012) to prepare individual bacmids. Individual bacmids were transfected into Sf9 cells to generate low titre P1 baculoviruses, which were subsequently used to prepare high titre P2 and P3 baculoviruses using the Bac to Bac system following the manufacturer’s recommendations (Invitrogen). For co-expression of the MRN complex, Sf9 cells (2.5 × 10⁶ per ml) grown in Sf-900 III SFM (Thermo Fisher Scientific, 12659017) were co-infected with baculoviruses expressing MRE11–Flag, RAD50–6×HIS and NBS1–Flag, and the complex was expressed at 27 °C for 48 h. Cells were collected by centrifugation at 500g for 15 min at 4 °C and the cell pellet was rinsed with PBS and stored at −80 °C until purification.

For expression and purification of human dynein complex mutants (or wild-type), mutations were engineered into the human dynein heavy chain (DHC)-containing plasmid, pbiG1a:6His-ZZ-SNAPf-DHC1. We used Gibson assembly to engineer point mutations – C1932S, R1962C, or both – into this plasmid. The PmeI-digested gene expression cassette from this plasmid was co-assembled with the PmeI-digested poly-gene cassette from pbiG1b:IC2/LIC2/Tctex1/Robl1/LC8 (encoding all dynein accessory chains) into PmeI-digested pbiG2ab using biGBac cloning strategies as previously described (Weissmann et al., 2016 (link)) (all wild-type plasmids were kind gifts from Andrew Carter). The final, sequence-verified plasmids (wild-type and mutant variants of pbiG2ab:6His-ZZ-SNAPf-DHC1/IC2/LIC2/Tctex1/Robl1/LC8) were used to generate recombinant baculoviral genomes by Tn7 transposition into DH10Bac cells (Life Technologies). White, PCR-confirmed colonies were inoculated into LB media supplemented with 7 µg/ml gentamycin, 10 µg/ml tetracycline and 50 µg/ml kanamycin and grown overnight at 37°C. Bacmid preparation was performed as described previously (Zhang et al., 2017 (link)), stored at 4°C, and used within 2 weeks for subsequent virus production (see below).