Softlink soft release avidin resin

Biotin-labeled oligonucleotides were synthesized by Sangon Biotech (for HLTF and KlRad5 co-precipitation) or Integrated DNA Technologies (for ScRad5 co-precipitation). A biotin-labeled dsDNA molecule was generated by heating a 5’-biotin-labeled self-annealing oligonucleotide D1 (Supplementary Table 4) to 95 °C followed by slow cooling to 4 °C. To characterize DNA binding to KlRad5 or HLTF, a 100 μl binding mixture containing the binding buffer, 2 μM KlRad5 or HLTF and 1 μM of the biotin-labeled dsDNA or dT₂₅ with 5’- or 3’-biotin labels was incubated with 10 μl SoftLink Soft Release avidin resin (Promega) for 1 h at 4 °C. The binding buffer contains 20 mM Tris (pH 7.5) and 50 mM sodium chloride unless indicated otherwise. After washing the resin three times with the binding buffer, bound DNA and protein were eluted with the binding buffer supplemented with 5 mM biotin and analyzed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). When indicated, SSB (Sangon Biotech) was added to a final concentration of 20 μM. To characterize DNA binding to ScRad5, 0.15 μM ScRad5 and 0.2 μM dT₂₅ with 5’- or 3’-biotin labels were incubated with 10 μl Streptavidin Magnetic Beads (NEB) in a 100 μl binding mixture with the same binding buffer. SSB was added to a final concentration of 1 μM when indicated.