Gene Ontology annotation through association of InterPro records with GO terms
Gene Ontology annotation based on Enzyme Commission mapping
Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
Electronic Gene Ontology annotations created by ARBA machine learning models
Combined Automated Annotation using Multiple IEA Methods
Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding.
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The pH dependence of the steady-state parameters shows that substrate binding requires protonation of a single residue, identified as Lys-83 by comparison with the K83A mutant, while turnover depends on a single deprotonation.
"We therefore conclude that protonation of Lys-83 in the wild type protein facilitates DNA binding."
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T5 5'-3' exonuclease belongs to the family of 5'-nucleases required for DNA replication and repair.
"T5 5'-3'-exonuclease is a member of a family of homologous 5'-nucleases essential for DNA replication and repair."
Roles of divalent metal ions in flap endonuclease-substrate interactions.
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The enzyme has two metal sites of different affinity; mutation of site I abolishes all activity, while mutation of the site II aspartates abolishes exonuclease action yet leaves structure-specific flap endonuclease activity intact.
"Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity."
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Metal stoichiometry selects the reaction: one ion in the high-affinity site supports endonucleolysis, two are needed for exonucleolysis, suggesting a two-metal mechanism for the latter.
"Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor."
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Divalent metal ions are obligatory cofactors for both of the enzyme's reactions, and site I binds cofactor about 30-fold more tightly than site II.
"Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II."
Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease.
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FENs, including the T5 enzyme, hydrolyse blunt-ended duplex DNA exonucleolytically and cleave 5'-bifurcated substrates endonucleolytically at the single/double-stranded junction.
"Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA."
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The phage enzyme was assayed on a defined substrate panel mimicking duplex, 5'-overhanging duplex, pseudo-Y, nicked and flap DNA, and cut most of them at multiple positions.
"The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated."
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Turnover of the T5 enzyme is limited by product release on every substrate tested.
"Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested."
Properties of overexpressed phage T5 D15 exonuclease. Similarities with Escherichia coli DNA polymerase I 5'-3' exonuclease.
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Homogeneous overexpressed D15 degrades both native and heat-denatured DNA, and shows no endonuclease activity on closed-circular or nicked duplex DNA.
"The purified enzyme is active on both native and heat-denatured DNA and shows no endonuclease activity on either double-stranded closed-circular or nicked DNA."
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The protein's mode of action resembles, without being identical to, the 5'-3' exonuclease activity of E. coli DNA polymerase I, with which its sequence is highly similar over the first 320 residues of the polymerase.
"The mode of action of this enzyme is similar to, although not identical to that of the 5'-3' exonuclease activity of E. coli DNA polymerase I."
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Oligonucleotide degradation depends on the hybridization state of the substrate as well as its sequence, an early indication of structure-dependent activity.
"The enzyme is also able to degrade some oligonucleotides in a manner which depends not only on the nucleotide sequence but also on the state of hybridization of the potential substrate."
Direct observation of DNA threading in flap endonuclease complexes.
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Crystal structures of T5 flap endonuclease with intact substrates and products show the single-stranded arm threaded through a hole enclosed by an inverted V-shaped helical arch over the active site.
"They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site."
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A series of complexes traces the reaction from free enzyme through pre-threading and fully threaded intermediates to a pseudo-product complex, supporting a fly-casting, thread, bend and barb mechanism.
"We have shown a series of snapshots in the FEN-catalyzed hydrolysis reaction from free enzyme to pseudo-product complex via pre-threading and fully threaded intermediates."
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The active-site aspartate variants D153K and D155K are hydrolytically inactive yet bind flap DNA more tightly than wild type, separating binding from catalysis.
"neither variant T5FenD153K nor a second variant, D155K, showed activity"
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A potassium ion bound by backbone carbonyls of the helix-three-turn-helix motif coordinates the DNA duplex, alongside the catalytic magnesium ions of the two active-site clusters.
"A potassium ion was bound via backbone carbonyl oxygens within this motif."
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The protein crystallised here is explicitly the gene D15 product of phage T5.
"also known as the gene D15 product"
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FEN-family enzymes are of applied as well as biological interest, the Taq polymerase 5'-nuclease being central to quantitative PCR and diagnostics.
"FEN homologs such as Taq polymerase are also central to real-time quantitative PCR and clinical diagnostics"
Requirement of a phage-induced 5'-exonuclease for the expression of late genes of bacteriophage T5.
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T5 amber mutants in gene D15 fail to express late genes: late proteins are virtually absent while an early enzyme is made at wild-type levels.
"Amber mutants of bacteriophage T5 defective in gene D15, which codes for a 5'-exonuclease, do not express late genes."
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The block is specific to late functions, with low lysozyme and serum-blocking power but normal synthesis of the early deoxyribonucleoside monophosphokinase.
"Synthesis of lysozyme and serum-blocking power is very low whereas the extent of synthesis of an early enzyme, deoxyribonucleoside monophosphokinase, is similar to that in wild-type infections."
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The authors' interpretation is indirect: the nuclease is thought to introduce gaps or nicks into T5 DNA so that late transcription can occur, rather than to act in transcription itself.
"It is proposed that one requirement for the expression of late T5 genes is the introduction of gaps or nicks in the T5 DNA so that late transcription can occur."
Isolation and characterization of a putative bacteriophage T5 transcription.replication enzyme complex from infected Escherichia coli.
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A roughly 5 MDa assembly isolated from T5-infected E. coli contains host RNA polymerase together with the phage proteins gpC2, gpD9, gpD5 and gpD15, and protects duplex DNA from nuclease.
"A well defined enzyme comples of approximately 5 X 10(6) daltons that contains phage and host cell components known to be required for the processes of phage transcription and DNA replication has been isolated from bacteriophage T5-infected Escherichia coli cells."
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gpD15 is named, with gpD5, as essential both for T5 DNA replication and for the initiation of late transcription.
"the proteins gpD5 (DNA-binding protein), and gpD15 (nuclease) which are both known to be essential for T5 DNA replication and for the initiation of late transcription"
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The authors read the composition as evidence that transcription and replication are integrated in the infected cell; the complex is presented as putative.
"The composition of the enzyme complex suggests that the processes of transcription and replication are integrated in T5-infected cells."
Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA.
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Maturation of replicated T5 DNA needs D15 for one specific step: unit-length single-stranded chains form without it, but the subsequent single-stranded fragments characteristic of virion DNA do not.
"The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease: however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional."
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Purified D15 nuclease, besides acting as a 5'-3' exonuclease, introduces endonucleolytic scissions into mature T5 DNA, but only where the duplex already carries single-stranded interruptions.
"the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, single-stranded interruptions"
Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.
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Binding measured by EMSA on a panel of defined duplexes is structure-selective: 5'-overhangs, flaps and pseudo-Y structures are bound, blunt duplexes and single-stranded oligonucleotides are not.
"However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease."
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No binding to blunt-ended duplexes or to single strands could be detected.
"Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA."
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Binding was deliberately assayed without the essential divalent metal cofactor, so it is separated from catalysis.
"The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor."
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The protein is defined as a 5'-3' exodeoxyribonuclease that additionally cleaves flap structures endonucleolytically, and neither of its cysteines is intimately involved in substrate binding.
"Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end)."
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.
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K83 is absolutely required for 5'-3' exonucleolytic activity on single-stranded DNA but dispensable for endonucleolytic cleavage of flap structures, separating the two activities of the same active site.
"Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures."
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The K83A crystal structure shows no significant perturbation around the metal sites, so the loss of exonuclease activity is not a folding or metal-binding artefact.
"Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site."
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K196 and K215 are not essential for either activity but contribute to substrate affinity, raising the dissociation constant from 5 nM to 200 nM (K196A) and 50 nM (K215A).
"Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A)."
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Wild-type enzyme has different pH optima for the endonuclease and exonuclease reactions, which with the mutant data indicates separate mechanisms.
"The wild-type protein has different pH optima for endonuclease and exonuclease activities."
A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins.
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A structure-specific single-cleavage assay on a 5'-overhanging hairpin substrate gives the steady-state parameters of the enzyme, whose catalytic acceleration of a specific phosphodiester bond is at least 10^15-fold.
"The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15)."
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All three of K83, K196 and K215 contribute to ground-state substrate binding, and K83 additionally contributes to the chemical step.
"Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency."
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The 5'-nuclease family to which this enzyme belongs has both exonucleolytic and structure-specific endonucleolytic activity and is conserved from bacteriophage to mammals.
"The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals."