Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure of a trapped endonuclease III-DNA covalent intermediate.

ABSTRACT: Nearly all cells express proteins that confer resistance to the mutagenic effects of oxidative DNA damage. The primary defense against the toxicity of oxidative nucleobase lesions in DNA is the base-excision repair (BER) pathway. Endonuclease III (EndoIII) is a [4Fe-4S] cluster-containing DNA glycosylase with repair activity specific for oxidized pyrimidine lesions in duplex DNA. We have determined the crystal structure of a trapped intermediate that represents EndoIII frozen in the act of repairing DNA. The structure of the protein-DNA complex provides insight into the ability of EndoIII to recognize and repair a diverse array of oxidatively damaged bases. This structure also suggests a rationale for the frequent occurrence in certain human cancers of a specific mutation in the related DNA repair protein MYH.

SUBMITTER: Fromme JC

PROVIDER: S-EPMC165637 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structure of a trapped endonuclease III-DNA covalent intermediate.

Fromme J Christopher JC Verdine Gregory L GL

The EMBO journal 20030701 13

Nearly all cells express proteins that confer resistance to the mutagenic effects of oxidative DNA damage. The primary defense against the toxicity of oxidative nucleobase lesions in DNA is the base-excision repair (BER) pathway. Endonuclease III (EndoIII) is a [4Fe-4S] cluster-containing DNA glycosylase with repair activity specific for oxidized pyrimidine lesions in duplex DNA. We have determined the crystal structure of a trapped intermediate that represents EndoIII frozen in the act of repai ...[more]

PMID: 12840008

Similar Datasets

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-A resolution.

Project description:2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

| S-EPMC31111 | biostudies-literature

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate.

Project description:Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

| S-EPMC125349 | biostudies-literature

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

Project description:The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.

| S-EPMC394490 | biostudies-other

Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I.

Project description:DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5' to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the -4 position 5' to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.

| S-EPMC3084087 | biostudies-literature

Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

Project description:The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.

| S-EPMC3499099 | biostudies-literature

DNA structure specificity of Rap endonuclease.

Project description:The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.

| S-EPMC148684 | biostudies-other

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.

Project description:Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.

| S-EPMC4505503 | biostudies-literature

Pso2 (SNM1) is a DNA structure-specific endonuclease.

Project description:Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures.

| S-EPMC3299998 | biostudies-literature

TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes

Project description:Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

| S-SCDT-10_1038-S44318-024-00169-3 | biostudies-other

Topoisomerase II-DNA complexes trapped by ICRF-193 perturb chromatin structure.

Project description:DNA topoisomerase II (topo II) is involved in unlinking replicating DNA and organizing mitotic chromosomes. Topo II is the target of many antitumour drugs. Topo II inhibition results in extensive catenation of newly replicated DNA and may potentially perturb chromatin assembly. Here, we show that the topo II inhibitor ICRF-193 does not prevent the bulk of nucleosome deposition, but perturbs nucleosome spacing in Xenopus egg extracts. This is due to the trapping of topo II-closed clamps on the DNA rather than increased DNA catenation. Inhibition of replicative DNA decatenation has in itself little or no effect on nucleosome deposition and spacing, suggesting that DNA can easily accommodate the sharp bending constraints imposed by the co-habitation of nucleosomes and catenane nodes. Chromatin perturbation by topo II clamps may explain some dominant cellular effects of ICRF-193. Nucleosome-driven bending of precatenane nodes may facilitate their unlinking by topo II during unperturbed replication.

| S-EPMC1369141 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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