Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation.

ABSTRACT: The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase ? (Pol ?), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol ?, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.

SUBMITTER: Dovrat D

PROVIDER: S-EPMC4191785 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Structure of monoubiquitinated PCNA: implications for DNA polymerase switching and Okazaki fragment maturation.

Project description:Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.

| S-EPMC3368864 | biostudies-literature

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation.

Project description:Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.

| S-EPMC3321178 | biostudies-literature

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins.

Project description:In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery.

| S-EPMC1156965 | biostudies-literature

Structural insight into Okazaki fragment maturation mediated by PCNA-bound FEN1 and RNaseH2

Project description:PCNA is a master coordinator of many DNA-metabolic events. During DNA replication, the maturation of Okazaki fragments involves at least four DNA enzymes, all of which contain PCNA-interacting motifs. However, the temporal relationships and functional modulations between these PCNA-binding proteins are unclear. Here, we developed a strategy to purify endogenous PCNA-containing complexes from native chromatin, and characterized their structures using cryo-EM. Two structurally resolved classes (PCNA-FEN1 and PCNA-FEN1-RNaseH2 complexes) have captured a series of 3D snapshots for the primer-removal steps of Okazaki fragment maturation. These structures show that product release from FEN1 is a rate-liming step. Furthermore, both FEN1 and RNaseH2 undergo continuous conformational changes on PCNA that result in constant fluctuations in the bending angle of substrate DNA at the nick site, implying that these enzymes could regulate each other through conformational modulation of the bound DNA. The structures of the PCNA-FEN1-RNaseH2 complex confirm the toolbelt function of PCNA and suggests a potential unrecognized role of RNaseH2, as a dsDNA binding protein, in promoting the 5'-flap cleaving activity of FEN1.

| S-SCDT-10_1038-S44318-024-00296-X | biostudies-other

Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly.

Project description:Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

| S-EPMC7195461 | biostudies-literature

High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication.

Project description:DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

| S-EPMC7817679 | biostudies-literature

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing.

Project description:During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

| S-EPMC9767926 | biostudies-literature

Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.

Project description:More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.

| S-EPMC8727043 | biostudies-literature

Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis.

Project description:Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5.-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3.-exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27Delta pol3-D520V mutants (defective for FEN1(RAD27) and the 3.-exonuclease of Pol delta) that produce long flaps and of dna2Delta mutants that are defective in cutting long flaps. On the contrary, pol32Delta or pif1Delta caused lethality of rad27Delta exo1Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.

| S-EPMC2590699 | biostudies-literature

Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation.

Project description:The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.

| S-EPMC4534484 | 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