Figure 7. Active CUL2^LRR1 is required for extraction of CMG components from chromatin during DNA replication termination in Xenopus egg extracts.

(a) Experimental scheme. (b) Replication reactions were performed in the presence of MLN4924 to stabilise CUL2^LRR1 on chromatin during DNA replication termination in mock-depleted extracts (treated with two rounds of IgG-beads). In contrast, neither CUL2 nor LRR1 were detected on chromatin in CUL2-depleted extracts, confirming the efficiency of the depletion. (c) Depletion of CUL2 also removes LRR1 from the extract (the panel shows immunoblots of the antibody-coupled beads after each of the two rounds of depletion). (d) Kinetics of DNA synthesis in extracts subjected to two rounds of immunoprecipitation with control IgG (‘mock depletion’) or with antibodies to Hs_CUL2-RBX1 (‘CUL2 depletion’, see Methods). Source data for repeats of this experiment are included in Supplementary Table 6. (e) In an analogous experiment, replication reactions were performed in ‘mock-depleted’ and CUL-depleted extracts. A pulse of α-dATP was added for 3’ at either the 60’ or 120’ timepoints, and the incorporation of radiolabel into nascent DNA was monitored after isolation of total DNA, indicating that replication proceeded and completed with similar kinetics in both extracts, consistent with the data in (d). (f) Kinetics of chromatin association of the indicated factors for the same experiment shown in (a-b). Note that the MCM7 immunoblot is over-exposed in order to reveal the ubiquitylated forms of the protein. (g) Mock-depleted or CUL2-depleted extracts were supplemented with the indicated recombinant proteins (X.l. LRR1, wt/mutant Hs_CUL2-RBX1 – see Methods), and chromatin was isolated from the 120’ timepoint in a similar experiment to that described above. Unprocessed scans of key immunoblots are shown in Supplementary Figure 8.