Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Crystal structures of ternary complexes of archaeal B-family DNA polymerases.

ABSTRACT: Archaeal B-family polymerases drive biotechnology by accepting a wide substrate range of chemically modified nucleotides. By now no structural data for archaeal B-family DNA polymerases in a closed, ternary complex are available, which would be the basis for developing next generation nucleotides. We present the ternary crystal structures of KOD and 9°N DNA polymerases complexed with DNA and the incoming dATP. The structures reveal a third metal ion in the active site, which was so far only observed for the eukaryotic B-family DNA polymerase ? and no other B-family DNA polymerase. The structures reveal a wide inner channel and numerous interactions with the template strand that provide space for modifications within the enzyme and may account for the high processivity, respectively. The crystal structures provide insights into the superiority over other DNA polymerases concerning the acceptance of modified nucleotides.

SUBMITTER: Kropp HM

PROVIDER: S-EPMC5718519 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Crystal structures of ternary complexes of archaeal B-family DNA polymerases.

Kropp Heike M HM Betz Karin K Wirth Johannes J Diederichs Kay K Marx Andreas A

PloS one 20171206 12

Archaeal B-family polymerases drive biotechnology by accepting a wide substrate range of chemically modified nucleotides. By now no structural data for archaeal B-family DNA polymerases in a closed, ternary complex are available, which would be the basis for developing next generation nucleotides. We present the ternary crystal structures of KOD and 9°N DNA polymerases complexed with DNA and the incoming dATP. The structures reveal a third metal ion in the active site, which was so far only obse ...[more]

PMID: 29211756

Similar Datasets

TT(N)mGCCTC inhibits archaeal family B DNA polymerases.

Project description:The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. However, thermostable proofreading DNA polymerases occasionally failed to amplify target fragment that could be amplified by Taq DNA polymerase. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. Here we showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. To the best of our knowledge, this is the first example of DNA sequence that could inhibit DNA polymerase in its single-stranded form.

| S-EPMC5792436 | biostudies-literature

A highly conserved family of inactivated archaeal B family DNA polymerases.

Project description:A widespread and highly conserved family of apparently inactivated derivatives of archaeal B-family DNA polymerases is described. Phylogenetic analysis shows that the inactivated forms comprise a distinct clade among archaeal B-family polymerases and that, within this clade, Euryarchaea and Crenarchaea are clearly separated from each other and from a small group of bacterial homologs. These findings are compatible with an ancient duplication of the DNA polymerase gene followed by inactivation and parallel loss in some of the lineages although contribution of horizontal gene transfer cannot be ruled out. The inactivated derivative of the archaeal DNA polymerase could form a complex with the active paralog and play a structural role in DNA replication.

| S-EPMC2527604 | biostudies-literature

Gene duplications in evolution of archaeal family B DNA polymerases.

Project description:All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria. Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases. We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S. solfataricus P2. The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S. solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains. We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae. It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S. solfataricus B3 ortholog. Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S. solfataricus P2 B3 and S. shibatae B3 paralogs are related to one of the two DNA polymerases of P. occultum. These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group. Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events.

| S-EPMC179013 | biostudies-other

Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Project description:Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

| S-EPMC2940834 | biostudies-literature

Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

Project description:Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and β4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.

| S-EPMC9064986 | biostudies-literature

Crystal Structures of Ternary Complexes of MEF2 and NKX2-5 Bound to DNA Reveal a Disease Related Protein-Protein Interaction Interface.

Project description:MEF2 and NKX2-5 transcription factors interact with each other in cardiogenesis and are necessary for normal heart formation. Despite evidence suggesting that these two transcription factors function synergistically and possibly through direct physical interactions, molecular mechanisms by which they interact are not clear. Here we determined the crystal structures of ternary complexes of MEF2 and NKX2-5 bound to myocardin enhancer DNA in two crystal forms. These crystal structures are the first example of human MADS-box/homeobox ternary complex structures involved in cardiogenesis. Our structures reveal two possible modes of interactions between MEF2 and NKX2-5: MEF2 and NKX bind to adjacent DNA sites to recognize DNA in cis; and MEF2 and NKX bind to different DNA strands to interact with each other in trans via a conserved protein-protein interface observed in both crystal forms. Disease-related mutations are mapped to the observed protein-protein interface. Our structural studies provide a starting point to understand and further study the molecular mechanisms of the interactions between MEF2 and NKX2.5 and their roles in cardiogenesis.

| S-EPMC8099449 | biostudies-literature

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.

Project description:Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase-DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.

| S-EPMC3575838 | biostudies-literature

Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes.

Project description:Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH.

| S-EPMC5119734 | biostudies-literature

Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation.

Project description:We discovered a thermostable enzyme from the archaeon Pyrococcus furiosus (Pfu), which increases yields of PCR product amplified with Pfu DNA polymerase. A high molecular mass (>250 kDa) complex with PCR-enhancing activity was purified from Pfu extracts. The complex is a multimer of two discrete proteins, P45 and P50, with significant similarity to bacterial dCTP deaminase/dUTPase and DNA flavoprotein, respectively. When tested in PCR, only recombinant P45 exhibited enhancing activity. P45 was shown to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate. Pfu dUTPase improves the yield of products amplified with Pfu DNA polymerase by preventing dUTP incorporation and subsequent inhibition of the polymerase by dU-containing DNA. dUTP was found to accumulate during PCR through dCTP deamination and to limit the efficiency of PCRs carried out with archaeal DNA polymerases. In the absence of dUTP inhibition, the combination of cloned Pfu DNA polymerase and Pfu dUTPase (PfuTurbo DNA polymerase) can amplify longer targets in higher yield than Taq DNA polymerase. In vivo, archaeal dUTPases may play an essential role in preventing dUTP incorporation and inhibition of DNA synthesis by family B DNA polymerases.

| S-EPMC117351 | biostudies-literature

New insights into the structures and interactions of bacterial Y-family DNA polymerases.

Project description:Bacterial Y-family DNA polymerases are usually classified into DinB (Pol IV), UmuC (the catalytic subunit of Pol V) and ImuB, a catalytically dead essential component of the ImuA-ImuB-DnaE2 mutasome. However, the true diversity of Y-family polymerases is unknown. Furthermore, for most of them the structures are unavailable and interactions are poorly characterized. To gain a better understanding of bacterial Y-family DNA polymerases, we performed a detailed computational study. It revealed substantial diversity, far exceeding traditional classification. We found that a large number of subfamilies feature a C-terminal extension next to the common Y-family region. Unexpectedly, in most C-terminal extensions we identified a region homologous to the N-terminal oligomerization motif of RecA. This finding implies a universal mode of interaction between Y-family members and RecA (or ImuA), in the case of Pol V strongly supported by experimental data. In gram-positive bacteria, we identified a putative Pol V counterpart composed of a Y-family polymerase, a YolD homolog and RecA. We also found ImuA-ImuB-DnaE2 variants lacking ImuA, but retaining active or inactive Y-family polymerase, a standalone ImuB C-terminal domain and/or DnaE2. In summary, our analyses revealed that, despite considerable diversity, bacterial Y-family polymerases share previously unanticipated similarities in their structural domains/motifs and interactions.

| S-EPMC6511836 | 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