Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure of the polymerase ? holoenzyme and atomic model of the leading strand replisome.

ABSTRACT: The eukaryotic leading strand DNA polymerase (Pol) ? contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ? holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ?-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ? revealing that the Dpb3-Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ? active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.

SUBMITTER: Yuan Z

PROVIDER: S-EPMC7308368 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome.

Yuan Zuanning Z Georgescu Roxana R Schauer Grant D GD O'Donnell Michael E ME Li Huilin H

Nature communications 20200622 1

The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε reveali ...[more]

PMID: 32572031

Similar Datasets

DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis.

Project description:DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.

| S-EPMC7228970 | biostudies-literature

CMG-Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome.

Project description:The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.

| S-EPMC5402455 | biostudies-literature

Dynamics of leading-strand lesion skipping by the replisome.

Project description:The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate-limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis.

| S-EPMC3877186 | biostudies-literature

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

Project description:DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol ?/primase initiates primers on both strands that are extended by Pol ? on the leading strand and by Pol ? on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ? for leading-strand synthesis, but to date a direct interaction between CMG and Pol ? has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ?. Using pure CMG and Pol ?, we reconstituted a stable 15-subunit CMG-Pol ? complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ? is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ?, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol ? function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ? on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ? and CMG provides an explanation for specific targeting of Pol ? to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

| S-EPMC4217400 | biostudies-literature

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme.

Project description:In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

| S-EPMC7863874 | biostudies-literature

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Project description:Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

| S-EPMC10711444 | biostudies-literature

The eukaryotic replisome tolerates leading-strand base damage by replicase switching.

Project description:The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low-fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading-strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase-polymerase uncoupling a switch from Pol ε, the canonical leading-strand replicase, to the lagging-strand replicase Pol δ, facilitates rapid, efficient and error-free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8-oxoguanine. We propose that replicase switching may promote continued leading-strand synthesis whenever the replisome encounters leading-strand damage that is bypassed more efficiently by Pol δ than by Pol ε.

| S-EPMC7917549 | biostudies-literature

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.

Project description:Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

| S-EPMC6265327 | biostudies-literature

Structure of the PolIII?-?c-DNA complex suggests an atomic model of the replisome.

Project description:The C-terminal domain (CTD) of the ? subunit of the clamp loader (?c) binds to both the DnaB helicase and the DNA polymerase III ? subunit (PolIII?), and determines their relative positions and orientations on the leading and lagging strands. Here, we present a 3.2 Å resolution structure of Thermus aquaticus PolIII? in complex with ?c and a DNA substrate. The structure reveals that the CTD of ?c interacts with the CTD of PolIII? through its C-terminal helix and the adjacent loop. Additionally, in this complex PolIII? displays an open conformation that includes the reorientations of the oligonucleotide-binding fold and the thumb domain, which may be an indirect result of crystal packing due to the presence of the ?c. Nevertheless, the position of the ?c on PolIII? allows us to suggest an approximate model for how the PolIII? is oriented and positioned on the DnaB helicase.

| S-EPMC3652607 | biostudies-literature

Collaborative coupling between polymerase and helicase for leading-strand synthesis.

Project description:Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.

| S-EPMC3401439 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data