Extended Data Fig. 2 |. Recombinant TRAIP supports CMG unloading at cisplatin-ICLs.

a, NPE immunodepleted of TRAIP was loaded alongside a dilution series of mock-depleted NPE and blotted for TRAIP. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected proteins are marked with asterisks.

b, Bacterially-expressed rTRAIP^WT, rTRAIP^R18C, His₆-SUMO-rTRAIP^WT, and His₆-SUMO-rTRAIP^ΔPIP (comprising residues 1-455) were partially purified, resolved by SDS-PAGE, and visualized with Coomassie Brilliant Blue staining. Note that His₆-SUMO-rTRAIP is obscured by co-migrating, contaminating proteins. Bacterially-expressed rTRAIP was used for all subsequent experiments, unless otherwise indicated.

c, rTRAIP^WT and rTRAIP^R18C were expressed in Sf9 insect cells and purified using an N-terminal 3×FLAG tag. The tag was then cleaved using 3C protease. The recombinant proteins, along with a buffer sample containing 3C protease only, were resolved by SDS-PAGE and visualized with Coomassie Brilliant Blue staining.

d, Mock- and TRAIP-depleted extracts supplemented with rTRAIP^WT or rTRAIP^R18C used in Fig. 1b were analyzed by immunoblotting for TRAIP. The absence of the non-specific bands seen in a may be due to shorter incubation with the TRAIP antibody. The concentration of added recombinant TRAIP relative to endogenous TRAIP fluctuates among experiments (e.g. compare panel d and Extended Data Fig. 3b). We ascribe this difference to variations in non-specific removal of endogenous TRAIP from extracts during the mock-depletion procedure, and possibly also in the delivery of recombinant TRAIP into extract.

e, pICL^Pt was replicated in mock- or TRAIP-depleted extracts supplemented with [α-³²P]dATP and Sf9-expressed rTRAIP^WT, rTRAIP^R18C, or 3C protease alone and analyzed as in Fig. 1b.

f, Extracts used in the replication reaction shown in e were analyzed as in d.

g, pICL^Pt was replicated in the indicated egg extracts with [α-³²P]dATP and analyzed as in Fig. 1b.

h, Extracts used in the replication reaction shown in g were analyzed as in d. Note that deleting the C-terminal PIP box disrupts the epitope for the TRAIP antibody used for immunoblotting. Therefore, to assess the activity of TRAIP^ΔPIP in ICL repair relative to TRAIP^WT, His₆-SUMO-tagged proteins were added back to TRAIP-depleted extract and assayed in g. The relative amounts of His₆-SUMO-TRAIP^WT and His₆-SUMO-TRAIP^ΔPIP were compared by detecting the histidine tag. By blotting the same extracts for TRAIP, a comparison of the relative concentrations of His₆-SUMO-TRAIP^ΔPIP and endogenous TRAIP was made.

i, Mock- and TRAIP-depleted extracts used in the replication reactions shown in Fig. 1c and d were analyzed as in d.