Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome

Patrik Eickhoff,Hazal B Kose,Fabrizio Martino,Tatjana Petojevic,Ferdos Abid Ali,Julia Locke,Nele Tamberg,Andrea Nans,James M Berger,Michael R Botchan,Hasan Yardimci,Alessandro Costa

doi:10.1016/j.celrep.2019.07.104

Abstract

SummaryIn the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.

Highlights

Chromosome duplication is catalyzed by the replisome, a multisubunit complex that combines DNA unwinding by the replicative helicase and synthesis by dedicated polymerases (Pellegrini and Costa, 2016)
The helicase function is provided by the Cdc45-minichromosome maintenance (MCM)-Go-Ichi-Ni-San (GINS) (CMG) assembly comprising Cdc45, GINS, and a hetero-hexameric motor known as the MCM complex (Ilves et al, 2010; Moyer et al, 2006)
How CMG activation promotes eviction of the lagging strand template from the MCM pore is unclear, it is known that extensive DNA unwinding requires replication protein A (RPA) (Douglas et al, 2018; Kose et al, 2019)

Summary

Introduction

Chromosome duplication is catalyzed by the replisome, a multisubunit complex that combines DNA unwinding by the replicative helicase and synthesis by dedicated polymerases (Pellegrini and Costa, 2016). The isolated CMG is a relatively slow helicase (Ilves et al, 2010), yet cellular rates of DNA replication can be achieved in vitro in the presence of fork-stabilization factors Csm3-Tof and Mrc (Yeeles et al, 2017). Despite these advances, a complete understanding of DNA fork unwinding and of fast and efficient replisome progression is still lacking (Abid Ali and Costa, 2016; Yeeles et al, 2017)

Results

Discussion

Conclusion