Figure 2. Replication fork structure inhibits CMG ubiquitylation by SCF^Dia2 and Cdc34.

(A) Purified proteins used in this study, analysed by SDS-PAGE and stained with colloidal Coomassie Blue. (B) The components described in (A) were incubated for 20’ at 30°C with the indicated concentrations of E3 (input), before isolation of CMG (IPs of Sld5) and immunoblotting of the indicated components. The asterisk indicates a small pool of free Mcm7 that was not targeted for ubiquitylation. (C) (i) Reaction scheme and illustration of the association of CMG with model DNA replication forks. Replisome components other than Ctf4 were omitted to limit DNA unwinding by CMG; (ii) Ubiquitylation reactions in the presence or absence of synthetic replication forks with the indicated 5’ flaps. (D) Reactions at the indicated [E3], plus or minus the model fork substrate with 15 nt 5’ flap from (C). (E) Reactions were performed as in (C), with addition of the indicated DNA (2 and 3 are the ssDNA oligos that were used to make 1). (F) Ubiquitylation reactions performed as in (D), in the absence of DNA and +/- E3 or CMG as indicated. See also Figure 2—figure supplements 1–2.

Figure 2—figure supplement 1. Reconstituted CMG ubiquitylation involves the conjugation of K48-linked ubiquitin chains to Mcm7.

(A) Reactions were performed as in Figure 2. The left panel presents the effects of dropping out the indicated components of the reactions. The right panel compares the indicated variants of ubiquitin (‘K0’=lysine free ubiquitin, ‘K48R’ has the indicated single change, whereas all lysines except K48 were mutated to arginine in ‘K48-only’). (B) Ubiquitylation of all 11 CMG components was monitored by immunoblotting under the indicated conditions (1 nM [E3]). (C) Similar reactions were performed in the presence of 25 nM [E3]. (D) Immunoblots of additional replisome components from the experiment in (B), together with longer exposure for Mcm4. (E) Immunoblots of additional replisome components from the experiment in (C).

Figure 2—figure supplement 2. Association of the CMG helicase with model DNA replication forks.

Recombinant CMG helicase was mixed with Cy3-labelled versions of the DNA substrates shown in Figure 2C (i), and then incubated for 60 min on ice. The CMG helicase was isolated by immunoprecipitation of Sld5, before immunoblotting and detection of Cy3 fluorescence.