Extended Data Fig. 5: TRAIP ubiquitylates numerous CMG subunits with heterotypically linked chains upon fork convergence at an ICL.

a, pICL-lacO^Pt was incubated with LacR before replication in mock- or TRAIP-depleted extracts. After 1 h of replication (to allow for termination of replication forks that do not converge on the ICL or lacO array as depicted in Figs. 2c), 0.3 mM NMS-873 was added and the extracts were incubated for 5 min to inhibit p97. IPTG (10 mM) and Sf9-expressed wild-type rTRAIP were then added as indicated and the extract was incubated for 2 h to disrupt LacR DNA binding and allow fork convergence and TRAIP-dependent ubiquitylation. The plasmid was recovered and analysed for the indicated proteins. Two independent experiments. b, The extracts described in a were analysed for TRAIP. Two independent experiments. c, pICL-lacO^Pt was incubated with LacR before replication in undepleted egg extracts. After 30 min of replication (to allow for termination of replication forks that do not converge on the ICL or lacO array as depicted in Figs. 2c), 0.3 mM NMS-873 was added and the extracts were incubated for 5 min to inhibit p97. IPTG (10 mM) was then added as indicated to disrupt LacR DNA binding and the extract was incubated for 1 h to allow for fork convergence. The plasmid was recovered and analysed for the indicated proteins. Two independent experiments. d, Recombinant CMG was purified, resolved by SDS–PAGE, and visualized with SYPRO Ruby staining. Results are typical of at least five independent purifications. e, rTRAIP ubiquitin ligase activity. Wild-type rTRAIP or rTRAIP(R18C) was combined with ubiquitin, E1 ligase, three E2 ligases (UbcH5a, UbcH5b, UbcH5c) and ATP as indicated. Polyubiquitin chain synthesis (top) and TRAIP autoubiquitylation (bottom) were detected by immunoblotting the reactions with ubiquitin and TRAIP antibody, respectively. rTRAIP(R18C) was much more compromised in forming free polyubiquitin chains in this assay than it was in ubiquitylating rCMG (see Fig. 2d). The data suggest that the interaction between TRAIP and CMG can suppress, to a great extent, the profound ubiquitylation defect of the R18C mutation. Three independent experiments. f, pCtrl-lacO and pICL-lacO^Pt were replicated in undepleted extract as in c and recovered. Samples were treated with the indicated DUBs and analysed for the indicated proteins. Three independent experiments. g, pCtrl-lacO and pICL-lacO^Pt pre-bound with LacR were replicated in undepleted extract as in c. At the time of IPTG addition to release the LacR array and allow for fork convergence, 100 µM recombinant ubiquitin (wild-type or various lysine-to-arginine mutants) was added to the extract (which contains around 8 µM endogenous ubiquitin) and incubated for 1 h. The plasmid was recovered and analysed for the indicated proteins. Three independent experiments. h, Extracts used in g were analysed for ubiquitin. Some ubiquitin mutants contain a di-ubiquitin species (marked with an asterisk). Whether this arises upon addition to the extract is unclear. Three independent experiments.