Extended Data Fig. 11: Supporting data for model for regulation of CMG ubiquitylation.

a, The MCM:Dia2^LRRinterface is occluded in the inactive Mcm2-7 double hexamer. The structure of the budding yeast Mcm2-7 double hexamer is shown (PDB: 5BK4 (ref. ³⁹)): one Mcm2-7 hexamer is displayed as a cartoon, the other as a surface. Double-stranded DNA is coloured orange. The positions of the N-tier (N) and C-tier (C) are labelled for each hexamer. Inset: focused view of the regions of Mcm2-7 involved in interaction with the Dia2 LRR domain, demonstrating the inaccessibility of these regions to Dia2 in the context of a double hexamer. b, Reaction scheme for experiment in panel c, to monitor the suppression of CMG ubiquitylation by DNA in the absence of the indicated proteins (top), which are predicted to interact with the excluded DNA strand during lagging strand synthesis. Pif1 was omitted to block fork convergence. DNase was included after the replication step to release the replisome from DNA, which triggers CMG ubiquitylation⁸. c, Reaction conducted as in panel b and analysed by SDS-PAGE and immunoblot. This experiment was repeated twice. d, Reaction scheme for experiment in panel e, to monitor the interaction of SCF^Dia2 with the replisome. e, Reaction conducted as in panel d and analysed by SDS-PAGE and immunoblot. * is rabbit IgG. This experiment was repeated twice. For gel source data, see Supplementary Fig. 1.