Taylor-Cross, E.; Jones, M. L.; Roske, J. J.; Fletcher, E. E.; Chan, I. X. R.; Yeeles, J. T. P.

Activation of the CMG (CDC45-MCM-GINS) helicase by MCM10 is a central unresolved step in DNA replication. DNA is melted within a dimeric CMG complex and one strand is expelled from each topologically closed MCM ring. How MCM10 drives this process is unclear owing to a lack of structural information. Here, by determining structures of yeast CMG-Mcm10 complexes, and of human CMG-Pol {epsilon} dimers assembled by DONSON and bound to MCM10 and the helicase activator RECQL4, we reveal a conserved mechanism of CMG helicase activation in eukaryotes. MCM10 targets CMG dimers through highly conserved and species-specific interactions, that in human also involve RECQL4. Our data indicate this arrangement allows MCM10 to stimulate DNA unwinding by CMG and to utilise the associated conformational changes to drive single-stranded DNA ejection between MCM2 and MCM5. This mechanism provides an explanation for synchronized activation of two CMG helicases at origins of bidirectional replication.