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

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

doi:10.1016/j.celrep.2019.07.104

Cited by 86 publications

(156 citation statements)

References 72 publications

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“…A recent cryo-EM structure of yeast CMG, where translocation on a fork DNA substrate is blocked by biotin-streptavidin barriers, exhibited a short stretch of dsDNA enclosed by the Nterminal zinc finger (ZF) protrusions of Mcm2-7 . In a similar assay, we showed that the N-terminal end of MCM in Drosophila CMG interacts with parental DNA at the fork in an analogous manner (Eickhoff et al, 2019) suggesting that the mode of helicase engagement with DNA is conserved between Drosophila and yeast. A possible exit route for the displaced lagging-strand template was proposed to be formed by cavities between MCM ZF protrusions (Eickhoff et al, 2019, Li andO'Donnell, 2018).…”

Section: Cmg Bypasses a Protein Directly Crosslinked To The Excluded mentioning

confidence: 81%

“…It is not clear whether the recent CMG structures in complex with fork DNA exhibiting dsDNA within the MCM ZF domains represent the helicase in an active fork unwinding state or a paused state (Eickhoff et al, 2019;Georgescu et al, 2017). Because CMG can unwind dsDNA much more efficiently with the help of RPA, a high-resolution structure of CMG while unwinding duplex DNA in the presence of RPA should further elucidate how the helicase engages with DNA in the eukaryotic replisome.…”

Section: Discussionmentioning

confidence: 99%

“…Together with our data demonstrating that duplex engagement by CMG severely slows it down during DNA unwinding, we favour a model in which CMG engaged with the fork by encircling parental duplex through MCM ZF domains reflects a stalled helicase. Accordingly, multiple different conformations of parental duplex with respect to the N-terminal region of MCM were observed in Drosophila CMG/fork DNA structure suggesting plasticity in duplex engagement by the helicase (Eickhoff et al, 2019).…”

Section: Cmg Bypasses a Protein Directly Crosslinked To The Excluded mentioning

confidence: 97%

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Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

Kose

Xie

Cameron

et al. 2019

Preprint

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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...

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Section: Cmg Bypasses a Protein Directly Crosslinked To The Excluded mentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Cmg Bypasses a Protein Directly Crosslinked To The Excluded mentioning

confidence: 97%

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Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

Kose

Xie

Cameron

et al. 2019

Preprint

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“…A complicating feature when trying to analyse CMG translocation is that, unlike the homohexameric helicases of simpler organisms, the MCM2-7 motor of the CMG is a hetero-hexamer of six related but distinct subunits (Bochman, Bell, & Schwacha, 2008). Indeed, biochemical measures of DNA unwinding by purified fly CMG showed that ATP binding and hydrolysis are not equally important at all MCM ring interfaces (Eickhoff et al, 2019;Ilves, Petojevic, Pesavento, & Botchan, 2010). Furthermore, biological evidence in yeast shows that the importance of DNA binding is different among MCM subunits (Lam et al, 2013;Ramey & Sclafani, 2014).…”

mentioning

confidence: 99%

“…Recent cryoEM analyses of yeast CMG have led to the proposal of alternative translocation mechanisms, based on 'pumpjack' or 'inchworm' movements of the N-and Ctier of the MCM ring (Abid Yuan et al, 2016). A recent structural study of the fly CMG in conditions of DNA-fork unwinding (Eickhoff et al, 2019) imaged four distinct states of the helicase; the states formed the basis for an asymmetric model of DNA unwinding that accounted for the different roles of the MCM2-7 subunits in translocation.…”

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confidence: 99%

CryoEM structures of human CMG - ATPγS - DNA and CMG - AND-1 complexes

Rzechorzek

Hardwick

Jatikusumo

et al. 2020

Preprint

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DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPgS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5′-end to MCM5 at the 3′-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPgS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one b-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.

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Myc and the Replicative CMG Helicase: The Creation and Destruction of Cancer

Reed

Alexandrow

2020

BioEssays

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Myc‐driven tumorigenesis involves a non‐transcriptional role for Myc in over‐activating replicative Cdc45‐MCM‐GINS (CMG) helicases. Excessive stimulation of CMG helicases by Myc mismanages CMG function by diminishing the number of reserve CMGs necessary for fidelity of DNA replication and recovery from replicative stresses. One potential outcome of these events is the creation of DNA damage that alters genomic structure/function, thereby acting as a driver for tumorigenesis and tumor heterogeneity. Intriguingly, another potential outcome of this Myc‐induced CMG helicase over‐activation is the creation of a vulnerability in cancer whereby tumor cells specifically lack enough unused reserve CMG helicases to recover from fork‐stalling drugs commonly used in chemotherapy. This review provides molecular and clinical support for this provocative hypothesis that excessive activation of CMG helicases by Myc may not only drive tumorigenesis, but also confer an exploitable “reserve CMG helicase vulnerability” that supports developing innovative CMG‐focused therapeutic approaches for cancer management.

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

Cited by 86 publications

References 72 publications

Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

CryoEM structures of human CMG - ATPγS - DNA and CMG - AND-1 complexes

Myc and the Replicative CMG Helicase: The Creation and Destruction of Cancer

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