DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

Costa, Alessandro; Renault, Ludovic; Swuec, Paolo; Petojevic, Tatjana; Pesavento, James J.; Ilves, Ivar; MacLellan-Gibson, Kirsty; Fleck, Roland A.; Botchan, Michael R.; Berger, James M.

doi:10.7554/elife.03273

Cited by 107 publications

(193 citation statements)

References 61 publications

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“…Our earlier work demonstrated that Pol e binds the C-tier of CMG and Pol α-primase binds the N-tier (17). The new DNA orientation reverses our previous proposal of replisome architecture (17) that was based on the opposite DNA polarity through the D. melanogaster CMG and the archaeal MCM (15,16). The N-tier ahead of the C-tier during CMG translocation has profound implications, not only for the replisome architecture but also for origin initiation.…”

Section: Significancementioning

confidence: 52%

“…As another means to assess the direction that DNA proceeds through CMG, we used nanopointer 20-mer dT oligonucleotides with SA bound to biotin at either the 3′ or 5′ end. This strategy was used in the study of D. melanogaster CMG, except the DNA was a partial duplex (15). In side view, the N-tier and C-tier of CMG are easily recognized.…”

Section: Resultsmentioning

confidence: 99%

“…The observation that the 3′ and 5′ biotinylated nanopointers bind opposite faces of CMG supports the expectation that DNA binds CMG with a preferred polarity. We also used a partial duplex having the same sequence as used in the D. melanogaster CMG study (15). This DNA has a 3′ 40-mer ssDNA of dT residues and a 20-mer duplex containing a biotin-SA conjugate.…”

Section: Resultsmentioning

confidence: 99%

“…DNA 20-mer dT oligonucleotides biotinylated on either the 3′ (A) or 5′ (B) terminus were incubated with CMG and then with SA. (C) A tailed dsDNA from a previous work (15). Biotin was on the 3′ end of the shorter strand.…”

Section: Resultsmentioning

confidence: 99%

“…High-resolution studies of MCM and CMG bound to their DNA substrates are lacking, but a negative-stain EM study of a streptavidin (SA)-tipped partial duplex bound to Drosophila melanogaster CMG (15), and FRET studies of an archaeal MCM (16), indicate that the C-tier is ahead and pulls the N-tier ring behind during translocation. This DNA orientation requires the DNA to pass through the helicase in the opposite direction compared with the SF3, SF4, and SF5 hexameric helicases.…”

Section: Significancementioning

confidence: 99%

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Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

Georgescu

Yuan

Bai

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

180

299

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The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol e below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle doublestranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.CMG helicase | DNA replication | DNA polymerase | origin initiation | replisome R eplicative helicases are hexameric rings in all domains of life (1-3). In bacteria and archaea, the replicative helicase is a homohexamer and encircles single-strand (ss) DNA at a replication fork. Some viral and phage replicative helicases are also ring-shaped hexamers, including bovine papilloma virus (BPV) E1, simian virus 40 (SV40) large T-antigen (T-Ag), and the T4 and T7 phage helicases. Unlike other replicative helicases, the eukaryotic replicative Mcm2-7 helicase is composed of six nonidentical but homologous Mcm subunits that become activated upon assembly with five accessory factors (Cdc45 and GINS tetramer) to form the 11-subunit CMG (Cdc45, Mcm2-7, GINS) (4-6). Numerous studies have outlined the process that forms CMG at origins in which the Mcm2-7 heterohexamer is loaded onto DNA as an inactive double hexamer in G1 phase, and becomes activated in S phase by several initiation proteins and cell-cycle kinases that assemble Cdc45 and GINS onto Mcm2-7 to form the active CMG helicases (7-9).Helicases assort into six superfamilies (SF1-SF6) based on sequence alignments (10). The SF1 and SF2 helicases are generally monomeric and the SF3-SF6 helicases are hexameric rings used in DNA replication and other processes. The bacterial SF4 and SF5 helicases contain RecA-based motors and translocate 5′-3′, whereas the eukarytic SF3 and SF6 helicases contain AAA+ (ATPases associated with diverse cellular activities)-based motors and translocate 3′-5′ (3, 10). Examples of well-studied hexameric helicases include t...

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Section: Significancementioning

confidence: 52%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

Georgescu

Yuan

Bai

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

180

299

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Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

2017

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The astonishing efficiency and accuracy of DNA replication has long suggested that refined rules enforce a single highly reproducible sequence of molecular events during the process. This view was solidified by early demonstrations that DNA unwinding and synthesis are coupled within a stable molecular factory, known as the replisome, which consists of conserved components that each play unique and complementary roles. However, recent single-molecule observations of replisome dynamics have begun to challenge this view, revealing that replication may not be defined by a uniform sequence of events. Instead, multiple exchange pathways, pauses, and DNA loop types appear to dominate replisome function. These observations suggest we must rethink our fundamental assumptions and acknowledge that each replication cycle may involve sampling of alternative, sometimes parallel, pathways. Here, we review our current mechanistic understanding of DNA replication while highlighting findings that exemplify multi-pathway aspects of replisome function and considering the broader implications.

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

O'Donnell

2018

BioEssays

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The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

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DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

Cited by 107 publications

References 61 publications

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

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

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