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.

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.

Summary Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.

The DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression. steric exclusion, a mechanism shared by all known replicative helicases (Egelman et al., 1995;Fu et al., 2011;Kaplan, 2000;Lee et al., 2014;Yardimci et al., 2012b).Using single-molecule magnetic-tweezers, we earlier found that individual Drosophila CMG complexes exhibit forward and backward motion while unwinding dsDNA (Burnham et al., 2019), similar to E1 and T7 gp4 helicases (Johnson et al., 2007;Lee et al., 2014;Syed et al., 2014). Furthermore, the helicase often enters long-lived paused states leading to an average unwinding rate of 0.1-0.5 base pairs per second (bps −1 ), which is approximately two orders of magnitude slower than eukaryotic replication fork rates observed in vivo (Anglana et al., 2003;Raghuraman et al., 2001). However, recent single-molecule work with yeast CMG suggests that the helicase translocates on ssDNA at 5-10 bps -1 (Wasserman et al., 2019). Single-molecule trajectories by other replicative helicases such as DnaB and gp4 suggest that helicase pausing during dsDNA unwinding is a general property of these enzymes (Ribeck et al., 2010;. However, it is not clear why replicative helicases frequently halt whilst moving at the fork and how higher speeds are achieved by the entire replisome.The rate of DNA unwinding by E.coli DnaB and T7 gp4 is substantially enhanced when engaged with their corresponding replicative polymerases (Kim et al., 1996;Stano et al., 2005) suggesting that rate of fork progression in eukaryotes may also depend on DNA synthesis.Likewise, uncoupling of CMG from the leading-strand polymerase leads to fork slowing in an in vitro purified yeast system (Taylor and Yeeles, 2019). 5-10 fold reduction in helicase speed was also observed in Xenopus egg extracts when DNA synthesis was inhibited by aphidicolin (Sparks et al., 2019). However, this decrease in CMG translocation rate is not sufficient to account for the approximate 100-fold lowe...