Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.

ABSTRACT: Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.

SUBMITTER: Bellendir SP

PROVIDER: S-EPMC5435919 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.

Project description:LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.

| S-EPMC6294521 | biostudies-literature

The importance of fitting in: conformational preference of selenium 2' modifications in nucleosides and helical structures.

Project description:Selenomethionine incorporation has proven useful in X-ray crystallography of proteins to obtain phase information. In nucleic acids, the introduction of selenium to different positions is beneficial for solving the phase problem as well, but its addition to the 2' position also significantly enhances the crystal formation. The selenium modification in a single nucleotide shows a preference towards 2'-endo sugar puckering, which is in conflict with existing crystal structures where the duplex incorporated 2'-selenium-modified nucleotide is exclusively found in a 3'-endo conformation. Our work provides a rationale why 2'-selenium modifications facilitate crystallization despite this contradictory behavior.

| S-EPMC4852287 | biostudies-literature

Population differences in host plant preference and the importance of yeast and plant substrate to volatile composition.

Project description:Divergent selection between environments can result in changes to the behavior of an organism. In many insects, volatile compounds are a primary means by which host plants are recognized and shifts in plant availability can result in changes to host preference. Both the plant substrate and microorganisms can influence this behavior, and host plant choice can have an impact on the performance of the organism. In Drosophila mojavensis, four geographically isolated populations each use different cacti as feeding and oviposition substrates and identify those cacti by the composition of the volatile odorants emitted. Behavioral tests revealed D. mojavensis populations vary in their degree of preference for their natural host plant. Females from the Mojave population show a marked preference for their host plant, barrel cactus, relative to other cactus choices. When flies were given a choice between cacti that were not their host plant, the preference for barrel and organ pipe cactus relative to agria and prickly pear cactus was overall lower for all populations. Volatile headspace composition is influenced by the cactus substrate, microbial community, and substrate-by-microorganism interactions. Differences in viability, developmental time, thorax length, and dry body weight exist among populations and depend on cactus substrate and population-by-cactus interactions. However, no clear association between behavioral preference and performance was observed. This study highlights a complex interplay between the insect, host plant, and microbial community and the factors mediating insect host plant preference behavior.

| S-EPMC5468138 | biostudies-literature

AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis.

Project description:DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3'-phosphor-?, ?-unsaturated aldehyde and 3'-phosphate by successive ?- and ?-eliminations, respectively. The kinetic studies revealed that these 3'-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.

| S-EPMC4191409 | biostudies-literature

Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate.

Project description:Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site.IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria.

| S-EPMC6879718 | biostudies-literature

Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.

Project description:Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

| S-EPMC4184034 | biostudies-literature

ECHDC1 knockout mice accumulate ethyl-branched lipids and excrete abnormal intermediates of branched-chain fatty acid metabolism.

Project description:The cytosolic enzyme ethylmalonyl-CoA decarboxylase (ECHDC1) decarboxylates ethyl- or methyl-malonyl-CoA, two side products of acetyl-CoA carboxylase. These CoA derivatives can be used to synthesize a subset of branched-chain fatty acids (FAs). We previously found that ECHDC1 limits the synthesis of these abnormal FAs in cell lines, but its effects in vivo are unknown. To further evaluate the effects of ECHDC1 deficiency, we generated knockout mice. These mice were viable, fertile, showed normal postnatal growth, and lacked obvious macroscopic and histologic changes. Surprisingly, tissues from wild-type mice already contained methyl-branched FAs due to methylmalonyl-CoA incorporation, but these FAs were only increased in the intraorbital glands of ECHDC1 knockout mice. In contrast, ECHDC1 knockout mice accumulated 16-20-carbon FAs carrying ethyl-branches in all tissues, which were undetectable in wild-type mice. Ethyl-branched FAs were incorporated into different lipids, including acylcarnitines, phosphatidylcholines, plasmanylcholines, and triglycerides. Interestingly, we found a variety of unusual glycine-conjugates in the urine of knockout mice, which included adducts of ethyl-branched compounds in different stages of oxidation. This suggests that the excretion of potentially toxic intermediates of branched-chain FA metabolism might prevent a more dramatic phenotype in these mice. Curiously, ECHDC1 knockout mice also accumulated 2,2-dimethylmalonyl-CoA. This indicates that the broad specificity of ECHDC1 might help eliminate a variety of potentially dangerous branched-chain dicarboxylyl-CoAs. We conclude that ECHDC1 prevents the formation of ethyl-branched FAs and that urinary excretion of glycine-conjugates allows mice to eliminate potentially deleterious intermediates of branched-chain FA metabolism.

| S-EPMC8473548 | biostudies-literature

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.

Project description:The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

| S-EPMC24846 | biostudies-literature

Structures of intermediates during RES complex assembly.

Project description:The action of the spliceosome depends on the stepwise cooperative assembly and disassembly of its components. Very strong cooperativity was observed for the RES (Retention and Splicing) hetero-trimeric complex where the affinity from binary to tertiary interactions changes more than 100-fold and affects RNA binding. The RES complex is involved in splicing regulation and retention of not properly spliced pre-mRNA with its three components--Snu17p, Pml1p and Bud13p--giving rise to the two possible intermediate dimeric complexes Pml1p-Snu17p and Bud13p-Snu17p. Here we determined the three-dimensional structure and dynamics of the Pml1p-Snu17p and Bud13p-Snu17p dimers using liquid state NMR. We demonstrate that localized as well as global changes occur along the RES trimer assembly pathway. The stepwise rigidification of the Snu17p structure following the binding of Pml1p and Bud13p provides a basis for the strong cooperative nature of RES complex assembly.

| S-EPMC4515762 | biostudies-literature

Substrate ectodomain is critical for substrate preference and inhibition of ?-secretase.

Project description:Understanding the substrate recognition mechanism of ?-secretase is a key step for establishing substrate-specific inhibition of amyloid ?-protein (A?) production. However, it is widely believed that ?-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that ?-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits A? production in a substrate-specific manner. Interestingly, these peptides suppress ?-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral A? levels. This report provides direct evidence of the substrate preference of ?-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-A? therapeutics to combat Alzheimer's disease.

| S-EPMC3826621 | 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