Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Probing the run-on oligomer of activated SgrAI bound to DNA.

ABSTRACT: SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.

SUBMITTER: Shah S

PROVIDER: S-EPMC4399878 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament.

Project description:Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

| S-EPMC6153303 | biostudies-literature

The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway.

Project description:Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.

| S-EPMC6153286 | biostudies-literature

New clues in the allosteric activation of DNA cleavage by SgrAI: structures of SgrAI bound to cleaved primary-site DNA and uncleaved secondary-site DNA.

Project description:SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg²(+) and bound to secondary-site DNA with either Mg²(+) or Ca²(+) are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca²(+) [Dunten et al. (2008), Nucleic Acids Res. 36, 5405-5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg²(+) structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3'-O leaving group.

| S-EPMC3016018 | biostudies-literature

Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI.

Project description:Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

| S-EPMC11367474 | biostudies-literature

Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry.

Project description:SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.

| S-EPMC6402485 | biostudies-literature

The structure of SgrAI bound to DNA; recognition of an 8 base pair target.

Project description:The three-dimensional X-ray crystal structure of the 'rare cutting' type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca(2+) bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn(2+), with strong binding at the protein-DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.

| S-EPMC2532715 | biostudies-literature

Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA.

Project description:Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (?65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions.

| S-EPMC5737210 | biostudies-literature

Probing helix propensity of monomers within a helical oligomer.

Project description:A simple strategy is proposed to assess the propensity of a given monomer to follow or not follow a particular helical scheme and to study helix reversal phenomena within helical oligomers. It consists of placing a monomer having a low helix propensity between two conformationally stable helical segments. Helix reversion then occurs preferentially at the site of this monomer, leading to the formation of isomers having P (right-handed) or M (left-handed) helicities at each of the two helical segments. The proportion between the P-P/M-M and P-M isomers is indicative of the stereochemical relations between the inserted monomer and the helical frame. Thus, xylylene or carboxylic acid anhydride spacers have been introduced between two helical oligoamides of 8-amino-2-quinolinecarboxylic acid. Both these spacers presumably lack some of the structural features that confer quinoline units with a high helix propensity. Only one species is observed in solution in the case of an anhydride spacer. This species was shown by x-ray crystallography to be a racemic mixture of P-P and M-M helices. Unexpectedly, the anhydride is consistently incorporated within helical oligoamides. For the xylylene spacer, the P-P/M-M racemate and P-M meso compound are in equal proportions in chloroform, showing that this spacer does not have a propensity to adopt any helical conformation in this solvent. However, the equilibria between the various isomers are shifted in toluene, where one species largely prevails. This species was shown by x-ray crystallography to be the P-P/M-M racemate. Molecular dynamics simulations are consistent with these solution data.

| S-EPMC1297517 | biostudies-literature

Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.

Project description:Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.

| S-EPMC3894315 | biostudies-literature

Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis.

Project description:We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers-exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the ?-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a ?-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.

| S-EPMC3000482 | 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