Figure 2. In vitro reconstitution of replisome‐specific ubiquitylation of CMG‐MCM‐7 by CUL‐2^LRR‐1 and an array of associated ubiquitylation enzymes.

1. Purified C. elegans proteins (see Materials and Methods).
2. Ubiquitylation of CMG‐MCM‐7 was reconstituted with the indicated factors as described in Materials and Methods. “Neddylation” indicates addition of the C. elegans ULA‐1_RFL‐1 E1 enzyme, the UBC‐12 E2 enzyme, the DCN‐1 E3 enzyme and NED‐8.
3. Equivalent reactions to those in (B) but using lysine‐free (K0) ubiquitin.
4. Similar reactions comparing wild‐type ubiquitin to the indicated ubiquitin mutants.
5. Reactions containing CMG and either CUL‐2^LRR‐1 or CUL‐2^VHL‐1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD‐5 and the indicated factors were monitored by immunoblotting.