The mechanism of eukaryotic CMG helicase activation

Max E Douglas,John F X Diffley,Ferdos Abid Ali,Alessandro Costa

doi:10.1038/nature25787

Abstract

The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.

Highlights

The initiation of eukaryotic DNA replication occurs in two discrete stages[1]: the minichromosome maintenance (MCM) complex is first loaded as a head-to-head double hexamer (DH) encircling duplex origin DNA during G1 phase; ‘firing factors’ convert each DH into two active CMG (Cdc45-MCM-GINS) helicases during S phase
These results show that CMG assembled from the DH can unwind DNA even when uncoupled from DNA synthesis, consistent with previous experiments[6,8]
CMG assembly and activation in the absence of RPA shifted a proportion of the 4 starting topoisomers (α+1, α, α-1, α-2) to a new set of supercoils, α-3, α-4, α-5 and α-6 indicating that each circle had been unwound by 3-4 helical turns (Figure 2a, lane 2)

Summary

Introduction

The initiation of eukaryotic DNA replication occurs in two discrete stages[1]: the minichromosome maintenance (MCM) complex is first loaded as a head-to-head double hexamer (DH) encircling duplex origin DNA during G1 phase; ‘firing factors’ convert each DH into two active CMG (Cdc45-MCM-GINS) helicases during S phase. Mcm[10] supported extensive unwinding and DNA synthesis even when added after CMG assembly was finished (Extended Data Figures 1a-d).

Results

Conclusion