Structral Study Of Eukaryotic DNA Replicative Helicase MCM

The accurate and complete replication of genomic DNA is essential for all life.DNA replication in eukaryotes is strictly regulated to ensure perpetuation of the integrity of the genome.To ensure that each replication origin is activated no more than once per cell division,the initiation of DNA replication is carried out in two temporally separated steps called “replication licensing” and “origin activation”.Replication licensing begins in early G1 phase when replicative DNA helicases are loaded around origins of DNA replication.The loaded helicases are then activated during S phase,with multiple helicase-activating factors involved.Eventually,DNA polymerase complexes and other accessory proteins are recruited to the bidirectional replication forks to form two active replisomes.The MCM(minichromosome maintenance)hexameric complex(Mcm2-7)forms the core of the eukaryotic replicative helicase.During G1 phase,two Cdt1–MCM heptamers are loaded onto each replication origin to form an inactive MCM double hexamer(DH)by the origin recognition complex(ORC)and Cdc6,but the detailed loading mechanism remains unclear.In the present study,we purify the yeast MCM hexamer and Cdt1–MCM heptamer,the two soluble precursors of the inactive chromatin bound DH from Saccharomyces cerevisiae and examine their structures by cryo-electron microscopy.Both complexes form left-handed coil structures with a 10-15 ? gap between Mcm5 and Mcm2 not wide enough for dsDNA to pass through and a central channel that is occluded by the CTD-WH motif of Mcm5.Cdt1 wraps around the N-terminal regions of Mcm2,Mcm6 and Mcm4,playing a role in stabilizing the whole complex.The intrinsic coiled structure of these precursors suggests that the planar double hexameric ring is a constrained high-energy structure consisting of two tightly coupled compressed springs.We propose that energy unleashed by the release of the springs provides the impetus for the initial unwinding of duplex DNA during DNA replication initiation.A repetitive spring-action mechanism is also consistent with the inchworm translocation mechanism proposed for the CMG helicase.