Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

Georgescu, Roxana E.; Schauer, Grant D.; Yao, Nina Y.; Langston, Lance D.; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; O'Donnell, Mike

doi:10.7554/elife.04988

Cited by 125 publications

(171 citation statements)

References 51 publications

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“…In Fig. 6B, reactions containing Pols α and e, but no Pol δ demonstrate that Pol e is incapable of Okazaki fragment extension, as shown in our earlier studies (10). The last three lanes that include Pols α, e, and δ in reactions containing 80 nM RFC show that Pol δ provides robust lagging strand synthesis, filling in the primed lagging strand fragments even at a high concentration of RFC (Fig.…”

Section: Quality Control Of Polmentioning

confidence: 74%

“…Pol α-primase contains both priming and DNA polymerase activities, and left unchecked, the polymerase activity of Pol α-primase has been demonstrated to function with CMG to replicate both the leading and lagging strands (9,10). Pol α-primase was earlier shown to function with the SV40 T-antigen helicase to synthesizes both the leading and lagging strands of the SV40 genome (35).…”

Section: Quality Control Of Polmentioning

confidence: 99%

“…These studies place Pol e on the leading strand and Pol δ on the lagging strand (34). Biochemical reconstitution of a functional leading-lagging strand eukaryotic replisome from pure proteins has been accomplished recently, enabling an independent method to assess the identity of polymerase assignment to specific DNA strands, as well as the mechanisms that underlie these assignments (9,10). Although polymerase recruitment mechanisms were expected based on bacterial studies, we did not anticipate the existence of quality control mechanisms in eukaryotes that remove polymerases that attach to the "wrong" strand of DNA.…”

Section: Quality Control Of Polmentioning

confidence: 99%

“…The intracellular ionic strength of S. cerevisiae is unknown, but at ionic strength under 70 mM, Pol δ-PCNA is highly processive for over 5 kb (13). Surprisingly, under these highly processive conditions Pol δ-PCNA exhibited poor and distributive activity with CMG on the leading strand (10). These paradoxical findings suggest that, in addition to recruitment mechanisms that place the proper polymerases on their respective strands, there also exist quality control mechanisms that remove improperly placed DNA polymerases on the wrong strand.…”

mentioning

confidence: 99%

“…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%

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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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Section: Quality Control Of Polmentioning

confidence: 74%

Section: Quality Control Of Polmentioning

confidence: 99%

Section: Quality Control Of Polmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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 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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Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours

Andrianova

Chetan

Sibin

et al. 2017

The Journal of Pathology

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Biallelic mismatch repair deficiency (bMMRD) in tumours is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1, and results in a characteristic mutational profile. In this article, we describe the genetic basis of ultramutated high-grade brain tumours in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high-grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant, p.R802*, in the PMS2 gene. Additionally, by genome sequencing of these tumours, we found extremely high somatic mutation rates (237/Mb and 123/Mb), as well as somatic mutations in the proofreading domain of POLE polymerase (p.P436H and p.L424V), which replicates the leading DNA strand. Most interestingly, we found, in both cancers, that the vast majority of mutations were consistent with the signature of POLE exo , i.e. an abundance of C>A and C>T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumour suppressor genes is more than two-fold lower in ultramutated tumours than in other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumours as being due to a combination of PMS2 germline and POLE somatic variants, and confirmed them as bMMRD/POLE exo disorders. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

Cited by 125 publications

References 51 publications

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

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

Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours

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