Fig. 5: Mutating the Mcm4 Walker B motif stabilises the Mcm ring.

a Schematic showing the pulldown assay used to assess ring splitting. Magnetic beads (brown spheres) were used to pulldown MCM2-7 using the HA-tag on Mcm3. During a high salt wash (H), ATP hydrolysis results in splitting of the ring at the Mcm4/Mcm7 interface in open-ring MCM2-7 (left). Closed ring-MCM2-7, such as double hexamers (DH), are stabilised by closure of the Mcm2/Mcm5 gate. The Mcm4 Walker B mutant (Mcm4-WB) prevents ATP hydrolysis at the Mcm4/Mcm7 interface, providing ring stability. b Pre-RC pulldown assay: WT MCM2-7 or Mcm4-WB were incubated in solution before being pulled-down via Mcm3. Under low salt conditions (L), the proteins maintain their hexameric forms. After a high-salt wash (H), MCM2-7 splits into Mcm2/4/6 and Mcm3/5/7 subcomplexes, with only Mcm3/5/7 being specifically eluted from the HA beads. Mcm4-WB maintains hexameric integrity. c Refeyn mass photometry of pulldown samples, washed with low salt buffer and then eluted using HA peptide. Trimer and hexamer peaks are visible for both WT MCM2-7 and Mcm4-WB. d Samples prepared as per part (c), but subjected to a high salt wash. Under these conditions, there is no detectable hexameric peak for WT MCM2-7. e Pulldown of Mcm proteins. After a high salt wash, ring splitting is observed in proteins lacking the Mcm4-WB mutation. f Pre-RC pulldown assay of proteins assembled on ARS1 DNA in solution. Under low salt conditions all Mcm proteins assemble pre-RC intermediates. After high salt washes hexameric MCM2-7 can be pulled down as a salt-stable DH. In complexes defective in ring closure, MCM2-7-ΔC5 and Mcm5-WH, trimeric protein is recovered. When combined with the Mcm4-WB mutation, the Mcm4/Mcm7 interface is stabilised, resulting in the elution of hexameric protein. Data are representative of three biological repeat and source data are provided as a Source Data file.