Extended Data Fig. 4 |. TRAIP and CRL2^LRR1 promote distinct CMG unloading pathways.

a, pCTRL was replicated in NPE in the presence of Geminin or the CDC7 inhibitor PHA-767491 (CDC7i). Eight minutes after addition of NPE, the plasmid was recovered and the indicated proteins were analyzed by immunoblot.

b, Cartoon depicting the effects of Geminin and CDC7i, and the step at which TRAIP is recruited to chromatin.

c, Extracts used in the replication reaction shown in Fig. 2e were blotted for TRAIP.

d, Top, upon termination of pCTRL replication, CMG (green) unloading depends on CRL2^LRR1-mediated MCM7 ubiquitylation (purple)^21,52, but it’s unknown whether unloading also requires TRAIP. Middle, CMGs that have converged at an ICL undergo TRAIP-dependent ubiquitylation (Fig. 1), but the involvement of CRL2^LRR1 is unknown. Bottom, if two origins fire on a single plasmid, one pair of replication forks converges at the ICL and undergoes TRAIP-dependent CMG unloading whereas a second pair undergoes CRL2^LRR1-dependent unloading. Because both pairs of CMGs should undergo ubiquitylation, in some experiments we include Culi to monitor only TRAIP-dependent ubiquitylation.

e, To determine whether TRAIP is required for CMG unloading during replication termination, we analyzed proteins associated with plasmids 60 min after replication initiation in mock- or TRAIP-depleted extracts containing p97i or Culi, as indicated. Chromatin was recovered and blotted for the indicated proteins. CMG unloading from pCTRL was unaffected by TRAIP depletion (compare lanes 4 and 7). Consistent with this, in the presence of p97i, TRAIP was not required for MCM7 ubiquitylation on pCTRL (compare lanes 2 and 5). In contrast, in the absence of TRAIP, CMG unloading from pICL^Pt was inhibited compared to the mock-depleted control (compare lanes 10 and 13), consistent with Fig. 1c. Similarly, TRAIP was essential for efficient MCM7 ubiquitylation on pICL^Pt (compare lanes 8 and 11, note the greater level of unmodified MCM7 in lane 11). The partial CMG unloading (lane 13) and residual MCM7 ubiquitylation observed on pICL^Pt in the absence of TRAIP (lane 11) were likely the result of termination events that occurred elsewhere on the plasmid (as described in d, bottom). Consistent with this interpretation, the combination of TRAIP depletion and Culi abolished MCM7 ubiquitylation (lane 12).

f, To determine whether CRL2^LRR1 contributes to CMG unloading at ICLs, pICL^Pt was replicated in undepleted extract containing p97i or Culi and analyzed as in Fig. 1b. Culi had no significant effect on the accumulation of Fast Figure 8 structures, consistent with CRL2^LRR1 being dispensable for CMG unloading at ICLs.

g, Left, to assess the effect of LRR1 depletion on CMG unloading at ICLs, NPE was immunodepleted of LRR1, loaded alongside a dilution series of mock-depleted NPE, and blotted for LRR1 and CUL2. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected protein is marked with an asterisk. Right, pICL^Pt was replicated in mock- or LRR1-depleted egg extracts and analyzed as in Fig. 1b. The absence of LRR1 had no effect on the formation of Fast Figure 8 structures, supporting the idea that CRL2^LRR1 is dispensable for CMG unloading at ICLs.

h, Nascent strand analysis of pICL^Pt replicating in mock- or LRR1-depleted extracts was performed as in Extended Data Fig. 3a. The CMG footprint disappeared with normal kinetics at the ICL in LRR1-depleted egg extract, consistent with CRL2^LRR1 not being required for CMG unloading at ICLs.