ATP Activity Assay of TRIP13 in the Presence of SHLD2L.3–REV7 Substrate.

The SHLD2L.3–REV7 dimer constructions containing REV7 mutations as well as TRIP13 with the E113A/E114A/E115A mutation (refers to polyE/A) were generated using QuikChange and confirmed by sequencing. The expression and purification of these mutants are the same as those for the wild-type proteins. A total of 2 μM of wild-type or mutated TRIP13 was incubated with 10 μM SHLD2L.3–REV7 substrates at 37 °C in 100 μL reaction buffer (20 mM Hepes pH 7.3, 100 mM NaCl, 5 mM MgCl₂, 10 mM ATP, and 1 mM DTT). As for indicated time points, 10 μL of reaction solution was taken out and added into a 384-well flat bottom white polystyrene microplate (Greiner Bio-One), and then mixed with 10 μL Kinase-Glo reagent (Promega) for 10 min at room temperature. The luminescence signals were measured using a TECAN Infinite M1000 reader with default mode (1,000 ms, no reduction). The data were analyzed with Prism 8 software (GraphPad).