Figure 5. Cdc48-Ufd1-Npl4 selectively unfold the ubiquitylated subunit(s) of CMG to drive replisome disassembly.

(A) CMG was ubiquitylated as in Figure 4 and then incubated for 20’ at 30°C in the presence or absence of Cdc48 as indicated. Ufd1-Npl4 was added to all samples. Subsequently, immunoprecipitations were performed with antibodies to the indicated factors, and the associated factors monitored by immunoblotting. (B) Fusion of Cdc48 to the bacterial FtsH protease generates a protein that specifically cleaves unfolded polypeptides that pass through the central channel of the Cdc48 hexamer. (C) Ubiquitylated CMG was immunoprecipitated with antibodies against Sld5, then incubated with Cdc48 (lanes 1 and 4), Cdc48 + FtsH (lanes 2 and 5) or Cdc48-FtsH fusion protein (lanes 3 and 6), all in the presence of Ufd1-Npl4 and ATP, before treatment for 60’ at 30°C with HsUSP2 deubiquitylase (lanes 4–6). Cleaved Mcm7 fragments were then detected by immunoblotting. (D) A similar reaction was performed as indicated and all 11 subunits of CMG were monitored by immunoblotting. See also Figure 5—figure supplement 1.

Figure 5—figure supplement 1. Ubiquitylated Mcm7 is unfolded during CMG helicase disassembly, and the ubiquitin chains must then be cleaved in order to release unfolded Mcm7 from Cdc48-Ufd1-Npl4.

(A) CMG was ubiquitylated as in Figure 4, before incubation 20’ at 30°C with the indicated factors. Ufd1-Npl4 was included in each case. Cdc48 and Cdc48-FtsH were included at 50 nM (+) or 200 nM (++). At the end of the reactions, the integrity of CMG was monitored by immunoprecipitation of Sld5. (B) Analogous CMG disassembly reactions to those in Figure 5D were performed in the presence of the indicated factors. At the end of the reactions, samples were incubated with HsUSP2, and cleavage of ubiquitylated Mcm4 and Mcm7 by Cdc48-FtsH was then detected by immunoblotting. (C) CMG disassembly was performed as in Figure 4A–B, before incubation with the indicated concentrations of the deubiquitylase Otu1 for 30’ at 30°C. Release of unfolded Mcm7 from Cdc48-Ufd1-Npl4 was then monitored by immunoprecipitation of Ufd1. (D) Quantification of the data in (C). The experiment was repeated three times, and the figure presents the mean values with standard deviations.