Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

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

doi:10.1038/nsmb.2851

Cited by 177 publications

(316 citation statements)

References 58 publications

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“…Omitting RFC-Δ1n or PCNA did not reduce repair in contrast to the previously observed complete dependence of Pol δ-dependent repair on RFC-Δ1n or PCNA (25). This result is consistent with prior observations that extensive DNA synthesis by Pol δ requires PCNA and its loading factor RFC, whereas extensive DNA synthesis by Pol e is not absolutely dependent on PCNA or RFC (54). Finally, omitting RPA did not reduce the amount of repair observed.…”

Section: Resultscontrasting

confidence: 37%

“…DNA replication is a highly coordinated process with leadingstrand DNA synthesis being performed by Pol e coupled to the cell division cycle 45 (Cdc45)/mini-chromosome maintenance proteins 2-7 (Mem2-7)/Sld5-Psf1-3 (GINS) DNA helicase complex (also called the CMG complex), and other proteins, and lagging-strand DNA synthesis primarily being performed by Pol δ and its accessory factor PCNA following initiation of laggingstrand synthesis by DNA Pol α/primase (52,54). MMR appears to be coupled to the replication fork as evidenced by the timing of MMR and the colocalization of the Msh2-Msh6 mispair recognition complex with replication fork proteins during S phase (55,56).…”

Section: Discussionmentioning

confidence: 99%

“…DNA Pol e was purified from S. cerevisiae cells using published overexpression plasmids/strains, buffers, and methods (54), with the following modifications: protease inhibitor cocktail (PIC) D and PIC W protease inhibitor mixtures were added to all buffers (25). Cells were grown to an OD 600 of 1.0 and protein expression was induced for 16 h before harvesting the cells and lysing them with seven passes through a Microfluidizer (Microfluidics).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

Bowen

Kolodner

2017

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

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Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5′ to 3′ excision by exonuclease 1 (Exo1) from a single-strand break 5′ to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3′ to the mispair, to make nicks 5′ to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase e (Pol e) may also play a role in MMR. Here we describe the reconstitution of Pol e-dependent MMR using S. cerevisiae proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze both short-patch and long-patch 5′ nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3′ to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol e was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol e can act in eukaryotic MMR in vitro.mutator phenotype | genome instability | DNA replication fidelity | DNA excision | DNA repair

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Section: Resultscontrasting

confidence: 37%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

Bowen

Kolodner

2017

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

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“…Thus, if there were an interaction between CMG and Pol δ, the affinity would be at least 3 orders of magnitude weaker than the interaction between CMG and Pol e. Therefore, CMG does not bind and stabilize Pol δ-PCNA. We therefore propose that Pol δ-PCNA undergoes collision release upon the encounter with CMG, accounting for the observed slow and distributive action of Pol δ-PCNA on the leading strand (9).…”

Section: Quality Control Of Polmentioning

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.…”

mentioning

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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Replication dynamics in fission and budding yeasts through DNA polymerase tracking

Vázquez

Antequera

2015

BioEssays

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The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions.

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Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

Cited by 177 publications

References 58 publications

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Replication dynamics in fission and budding yeasts through DNA polymerase tracking

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