Browse
Submit Data
Databases
API
Help

Dataset Information

34 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ?.

ABSTRACT: DNA polymerase ? (Pol?) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Pol? from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Pol?, Pol?, and Pol?. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Pol? is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.

SUBMITTER: Jain R

PROVIDER: S-EPMC4061903 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ε.

Jain Rinku R Vanamee Eva S ES Dzikovski Boris G BG Buku Angeliki A Johnson Robert E RE Prakash Louise L Prakash Satya S Aggarwal Aneel K AK

Journal of molecular biology 20131019 2

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Po ...[more]

PMID: 24144619

Similar Datasets

The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.

Project description:DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. Recently it was shown that the catalytic domain of yeast Polε (PolεCD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we show that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains significantly lower level of iron. The iron content of purified hPolECD samples correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding as well as DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. The binding studies demonstrated low affinity of hPolεCD to DNA and a strong effect of salt concentration on stability of the hPolεCD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s-1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 µM.

| S-EPMC9581978 | biostudies-literature

Crystal structure of yeast DNA polymerase ε catalytic domain.

Project description:DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.

| S-EPMC3986358 | biostudies-literature

Human DNA polymerase delta requires an iron-sulfur cluster for high-fidelity DNA synthesis.

Project description:Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron-sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data hence suggest that the FeS cluster in human Pol δ is an important co-factor that despite its C-terminal location has an impact on both DNA polymerase and exonuclease activities, and can influence the fidelity of DNA synthesis.

| S-EPMC6613617 | biostudies-literature

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Project description:Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo-, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo- on DNA-modified electrodes, a large cathodic signal centered at -140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo-, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo- bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo-. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo- via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

| S-EPMC8499023 | biostudies-literature

Strand displacement synthesis by yeast DNA polymerase ε.

Project description:DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.

| S-EPMC5041465 | biostudies-literature

Translesion synthesis of abasic sites by yeast DNA polymerase epsilon.

Project description:Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase epsilon bypasses a model abasic site with comparable efficiency to Pol eta and Dpo4, two translesion polymerases. DNA polymerase epsilon also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase epsilon with 3'-5' proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.

| S-EPMC2797225 | biostudies-literature

Mitochondria export iron-sulfur and sulfur intermediates to the cytoplasm for iron-sulfur cluster assembly and tRNA thiolation in yeast.

Project description:Iron-sulfur clusters are essential cofactors of proteins. In eukaryotes, iron-sulfur cluster biogenesis requires a mitochondrial iron-sulfur cluster machinery (ISC) and a cytoplasmic iron-sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [35S]cysteine to detect cytoplasmic Fe-35S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe-S)int, needed for cytoplasmic iron-sulfur cluster assembly. The mitochondrial biosynthesis of (Fe-S)int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe-S)int via the Atm1 transporter in the inner membrane, and we detected (Fe-S)int in active form. When (Fe-S)int was added to cytoplasm, CIA utilized it for iron-sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron-sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe-S)int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron-sulfur cluster synthesis. Of note, the (Fe-S)int is distinct from the sulfur intermediate called Sint, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron-sulfur cluster assembly in yeast cells.

| S-EPMC6579456 | biostudies-literature

Human DNA polymerase ε is phosphorylated at serine-1940 after DNA damage and interacts with the iron-sulfur complex chaperones CIAO1 and MMS19.

Project description:We describe a dynamic phosphorylation on serine-1940 of the catalytic subunit of human Pol ε, POLE1, following DNA damage. We also describe novel interactions between POLE1 and the iron-sulfur cluster assembly complex CIA proteins CIAO1 and MMS19. We show that serine-1940 is essential for the interaction between POLE1 and MMS19, but not POLE1 and CIAO1. No defect in either proliferation or survival was identified when POLE1 serine-1940 was mutated to alanine in human cells, even following treatment with DNA damaging agents. We conclude that serine-1940 phosphorylation and the interaction between serine-1940 and MMS19 are not essential functions in the C terminal domain of the catalytic subunit of DNA polymerase ε.

| S-EPMC4917431 | biostudies-literature

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

Project description:Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.

| S-EPMC4583046 | biostudies-literature

The in vitro fidelity of yeast DNA polymerase δ and polymerase ε holoenzymes during dinucleotide microsatellite DNA synthesis.

Project description:Elucidating the sources of genetic variation within microsatellite alleles has important implications for understanding the etiology of human diseases. Mismatch repair is a well described pathway for the suppression of microsatellite instability. However, the cellular polymerases responsible for generating microsatellite errors have not been fully described. We address this gap in knowledge by measuring the fidelity of recombinant yeast polymerase δ (Pol δ) and ɛ (Pol ɛ) holoenzymes during synthesis of a [GT/CA] microsatellite. The in vitro HSV-tk forward assay was used to measure DNA polymerase errors generated during gap-filling of complementary GT(10) and CA(10)-containing substrates and ∼90 nucleotides of HSV-tk coding sequence surrounding the microsatellites. The observed mutant frequencies within the microsatellites were 4 to 30-fold higher than the observed mutant frequencies within the coding sequence. More specifically, the rate of Pol δ and Pol ɛ misalignment-based insertion/deletion errors within the microsatellites was ∼1000-fold higher than the rate of insertion/deletion errors within the HSV-tk gene. Although the most common microsatellite error was the deletion of a single repeat unit, ∼ 20% of errors were deletions of two or more units for both polymerases. The differences in fidelity for wild type enzymes and their exonuclease-deficient derivatives were ∼2-fold for unit-based microsatellite insertion/deletion errors. Interestingly, the exonucleases preferentially removed potentially stabilizing interruption errors within the microsatellites. Since Pol δ and Pol ɛ perform not only the bulk of DNA replication in eukaryotic cells but also are implicated in performing DNA synthesis associated with repair and recombination, these results indicate that microsatellite errors may be introduced into the genome during multiple DNA metabolic pathways.

| S-EPMC3121764 | 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