CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; O’Donnell, Michael

doi:10.1073/pnas.1418334111

Cited by 144 publications

(174 citation statements)

References 43 publications

(86 reference statements)

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“…However, this new model contradicts much of the available data and is highly controversial (Burgers et al 2016;Lujan et al 2016). Most previous experimental studies confirm the classical model, including experiments with mutant polymerases (Pursell et al 2007;Kunkel and Burgers 2008;Nick McElhinny et al 2008;Johnson et al 2015) or incorporation of ribonucleotides Clausen et al 2015;Johnson et al 2015), where specific mutations were observed on the corresponding strands; experiments investigating the association of proteins with leading and lagging strands of DNA replication forks (Yu et al 2014); and biochemical experiments of assembly and stabilization of replication complexes (Georgescu et al , 2015Langston et al 2014). Moreover, recent evidence suggests that Pol epsilon does not proofread errors made by Pol delta (Flood et al 2015).…”

Section: Discussionmentioning

confidence: 99%

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

et al. 2017

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Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Differences in correction of errors produced during replication of the leading and the lagging DNA strands were reported in yeast and in human cancers, but the causes of these differences remain unclear. Here, we analyze data on human cancers with somatic mutations in two of the major DNA polymerases, delta and epsilon, that replicate the genome. We show that these cancers demonstrate a substantial asymmetry of the mutations between the leading and the lagging strands. The direction of this asymmetry is the opposite between cancers with mutated polymerases delta and epsilon, consistent with the role of these polymerases in replication of the lagging and the leading strands in human cells, respectively. Moreover, the direction of strand asymmetry observed in cancers with mutated polymerase delta is similar to that observed in MMR-deficient cancers. Together, these data indicate that polymerase delta (possibly together with polymerase alpha) contributes more mismatches during replication than its leading-strand counterpart, polymerase epsilon; that most of these mismatches are repaired by the MMR system; and that MMR repairs about three times more mismatches produced in cells during lagging strand replication compared with the leading strand.

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Section: Discussionmentioning

confidence: 99%

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

et al. 2017

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“…We also examined the effect of CMG on Pol e activity on RPA-coated PCNA-primed ssDNA, to determine whether CMG may directly inhibit Pol e. Unexpectedly, the result showed instead that CMG stimulates the rate of Pol e on an RPA-coated PCNA-primed ssDNA ( Fig. S8), from which we infer that formation of the previously documented CMGE complex (11,24) enhances Pol e enzymatic activity even in the absence of helicase activity.…”

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confidence: 81%

“…The replication protein A (RPA) heterotrimer binds and protects the single-strand (ss) DNA of the lagging strand against nucleases, and helps melt secondary structure in ssDNA. Reconstitution of the core eukaryotic replisome using all three replicative DNA polymerases has recently been accomplished with pure recombinant proteins in the S. cerevisiae system (9)(10)(11). These in vitro studies revealed that Pol e binds directly to CMG, forming a CMGE complex (a complex of CMG bound to Pol epsilon), and this recruits Pol e to PCNA on the leading strand for efficient extension.…”

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confidence: 99%

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Schauer

O'Donnell

2017

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

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The eukaryotic genome is primarily replicated by two DNA polymerases, Pol e and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol e and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol e on the lagging strand, and CMG protects Pol e against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol e binds CMG with a K d value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.D uplication of genetic material is performed by a dynamic interplay of numerous different proteins, collectively referred to as the replisome, that orchestrate their actions to accomplish efficient, high-fidelity replication of both the leading and lagging strands (1-3). Eukaryotes possess several replisome factors that have no homologs in bacteria, and also require two different DNA polymerases for bulk leading and lagging strand synthesis, and Escherichia coli and its phages use identical copies of a DNA polymerase for both strands of the duplex (4). Thus far, the evolutionary purpose behind the additional complexity in eukaryotes is unclear.At the heart of the eukaryotic replisome is an 11-subunit helicase complex referred to as CMG (Cdc45-Mcm2-7-GINS) (5-7). Eukaryotes use three different multisubunit B-family DNA polymerases (Pol) for replication. Pol e and Pol δ copy the bulk of the genome, and Pol α-primase generates hybrid RNA-DNA primers of about 25 nucleotides and, unlike Pol e and Pol δ, it lacks 3′-5′ exonuclease (proofreading) activity. The replication factor C (RFC) clamp loader is used to load PCNA (proliferating cell nuclear antigen) clamps onto primed sites that endow the replicative polymerases with high processivity (8). The replication protein A (RPA) heterotrimer binds and protects the single-strand (ss) DNA of the lagging strand against nucleases, and helps melt secondary structure in ssDNA. Reconstitution of the core eukaryotic replisome using all thre...

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“…S. cerevisiae CMG and Pol e were purified as previously described (46,47). To prepare EM grids, we first diluted each sample with 20 mM Tris-acetate, pH 7.5, 40 mM K-glutamate, 2 mM DTT, and 0.1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

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.

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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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CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Cited by 144 publications

References 43 publications

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

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

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