The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

H, Li; O'Donnell, Mike

doi:10.1002/bies.201700208

Cited by 72 publications

(62 citation statements)

References 99 publications

(247 reference statements)

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“…The key mechanism for establishing a stable association of replisomes with the replication forks is the maintenance of DNA in the hole of the ring‐shaped structure of replicative helicases. Replicative helicases, including gp4 in bacteriophage T7 , DnaB in Escherichia coli , MCM homo‐hexamer–Cdc45/RecJ‐like protein–GINS complex in archaea , and Cdc45, Mcm2–7 hexamer and GINS complex (CMG complex) in eukaryotes , have a ring‐like shape and are considered to hold unwound ssDNA in the hole of the ring . The helicases bound stably to DNA associate with DNA polymerase, which also binds to unwound ssDNA for DNA synthesis and interacts with a clamp complex, the β clamp in E. coli , or PCNA in eukaryotes.…”

Section: Stable Association Between the Replication Fork And Replisomementioning

confidence: 99%

Replication fork pausing at protein barriers on chromosomes

Hizume

Araki

2019

FEBS Letters

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When a cell divides prior to completion of DNA replication, serious DNA damage may occur. Thus, in addition to accuracy, the processivity of the replication forks is important. DNA synthesis at replication forks should be completed in time, and forks overcome aberrant structures on the template DNA, including damaged sites, using trans‐lesion synthesis, occasionally introducing mutations. By contrast, the protein barrier built on the DNA is known to block the progression of replication forks at specific chromosomal loci. Such protein barriers avert any collision of replication and transcription machineries, or control the recombination of specific loci. The components and the mechanisms of action of protein barriers have been revealed mainly using genetic and biochemical techniques. In addition to proteins involved in replication fork pausing, the interaction of the replicative helicase and DNA polymerase is also essential for replication fork pausing. Here, we provide an overview of replication fork pausing at protein barriers.

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Section: Stable Association Between the Replication Fork And Replisomementioning

confidence: 99%

Replication fork pausing at protein barriers on chromosomes

Hizume

Araki

2019

FEBS Letters

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“…The C-tier contains the motors with ATP sites located at subunit interfaces (Davey et al, 2003;Lyubimov et al, 2011). The ATP binding site of bacterial helicases is based on the RecA fold, while eukaryotic helicase ATP sites are based on the AAA+ fold (Erzberger and Berger, 2006;Li and O'Donnell, 2018;Lyubimov et al, 2011;O'Donnell and Li, 2018). While bacterial replicative helicases travel 5'-3' with the C-tier motors leading the way, eukaryotic helicases track 3'-5' on DNA with the N-face in front, pushed by motors in the C-tier (Enemark and Joshua-Tor, 2008;Li and O'Donnell, 2018;O'Donnell and Li, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…The ATP binding site of bacterial helicases is based on the RecA fold, while eukaryotic helicase ATP sites are based on the AAA+ fold (Erzberger and Berger, 2006;Li and O'Donnell, 2018;Lyubimov et al, 2011;O'Donnell and Li, 2018). While bacterial replicative helicases travel 5'-3' with the C-tier motors leading the way, eukaryotic helicases track 3'-5' on DNA with the N-face in front, pushed by motors in the C-tier (Enemark and Joshua-Tor, 2008;Li and O'Donnell, 2018;O'Donnell and Li, 2018). The way in which helicases engage ssDNA in the motor domains has been documented for several different hexameric replicative helicases and involve binding to loops in the ATPase domains (Abid Ali et al, 2017;Enemark and Joshua-Tor, 2006;Gao et al, 2019;Itsathitphaisarn et al, 2012;Li and O'Donnell, 2018;Meagher et al, 2019;O'Donnell and Li, 2018;Skordalakes and Berger, 2006;Thomsen and Berger, 2009).…”

Section: Introductionmentioning

confidence: 99%

DNA unwinding mechanism of a eukaryotic replicative CMG helicase

Yuan

Georgescu

Bai

et al. 2019

Preprint

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High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understand the unwinding mechanism. The eukaryotic replicative CMG helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the OB fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" for steric exclusion unwinding. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.

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“…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). If the displaced strand exits through a narrow opening between MCM ZF domains during unwinding, CMG is expected to pause when colliding with a bulky obstacle on this strand.…”

Section: Cmg Bypasses a Protein Directly Crosslinked To The Excludedmentioning

confidence: 99%

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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The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

Cited by 72 publications

References 99 publications

Replication fork pausing at protein barriers on chromosomes

Replication fork pausing at protein barriers on chromosomes

DNA unwinding mechanism of a eukaryotic replicative CMG helicase

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

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