Fig. 7
Composition of the replicative helicase productively engaged to the replication fork. a Diagram of the EM-based translocation assay. Multiple MCM double hexamers are loaded onto a linear stretch of duplex DNA capped with protein–DNA roadblocks. Only certain MCM double hexamers are activated and translocating CMGs push MCM double hexamers against the DNA roadblock. b Representative negative-stain micrograph reveals accumulation of MCM trains (marked with a khaki line) alongside isolated CMGs (marked with purple circles). Scale bar 50 nm. c Detail of a negative-stain micrograph revealing that MCM trains are made of stacked double hexamers loaded onto DNA. d Two-dimensional averages of the CMG capping the trains and opposed end of trains capped by a protein roadblock. Most MCM-pushing CMG particles are bound to Pol epsilon. e 2D averages of CMG-Pol epsilon and MCM double hexamers, next to a cartoon of an MCM blocked by a protein–DNA roadblock. f Atomic structures of CMG-Pol epsilon and MCM double hexamer, both bound to DNA. g 2D class averages of isolated CMGs observed in the MCM train experiment. Most CMGs that failed to translocate up to the roadblock are not engaged by Pol epsilon. h Cryo-electron tomogram of an MCM train. Scale bar 50 nm. i MCM and CMG structures placed into the cryo-electron tomogram using template matching approaches. This experiment supports the notion that CMG-Pol epsilon particles push MCM double hexamers against a protein–DNA roadblock to form MCM trains
