Gene Ontology annotation through association of InterPro records with GO terms
Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
Combined Automated Annotation using Multiple IEA Methods
UniProtKB entry P03680 (DPOL_BPPH2), DNA polymerase
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UniProt's curated FUNCTION line records three distinct enzymatic activities in the one chain, with experimental evidence: DNA polymerisation, terminal protein deoxynucleotidylation, and 3'-5' exonuclease.
"This polymerase possesses three enzymatic activities: DNA synthesis (polymerase), primer terminal protein (TP) deoxynucleotidylation, which is the formation of a covalent linkage (phosphoester) between the hydroxyl group of a specific serine residue in TP and 5'-dAMP, a reaction directed by the second T at the 3' end, and 3' to 5' exonuclease activity"
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The exonuclease has a proofreading purpose, and the two active sites are used without the primer leaving the enzyme.
"Exonuclease activity has a proofreading purpose"
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The entry places the protein in the type-B DNA polymerase family, the curated classification that the family-B InterPro signatures and the GO:0003887 rows rest on.
"Belongs to the DNA polymerase type-B family."
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Mg2+ is the curated cofactor, with four experimental references, and six Mg2+ ligand residues are mapped as BINDING features.
"Name=Mg(2+); Xref=ChEBI:CHEBI:18420;"
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The catalytic domains are separated along the chain: the N-terminal 191 residues carry the exonuclease and the strand displacement ability, residues 230-562 the initiation, polymerisation and pyrophosphorolytic activities.
"The N-terminus contains the 3'-5' exonuclease activity and strand displacement ability"
Bacillus subtilis phage phi29. Characterization of gene products and functions.
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Mutants in six phi29 cistrons fail to synthesise phage DNA; gene 2 is one of them. This is the basis of the CACAO GO:0039693 row, and the same paper is cited on CACAO rows for phi29 genes 1, 3, 6 and 17 for the same term.
"Mutants in six genes do not synthesize phage DNA."
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The paper's purpose was mapping cistrons to protein products, which is why it is cited across so many phi29 entries; it also identifies a lysis cistron, the basis of the CACAO rows on genes 4 and 15 for viral release by cytolysis.
"The protein products of 10 out of the 17 cistrons detected in the genome of phage phi29 have been identified."
Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex.
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Formation of the p3-dAMP initiation complex in extracts is absolutely dependent on a functional gene 2 product, with p3 and the phi29 DNA-protein p3 template; this is the mutant-based evidence behind both CACAO GO:0006270 rows.
"The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template."
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The survey is differentiating rather than blanket: the products of genes 5, 6 and 17 are not needed for the initiation step, even though their mutants are DNA-synthesis-defective.
"The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex."
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Host DNA polymerase III, gyrase and RNA polymerase inhibitors do not affect initiation, so the phage initiation machinery is autonomous of the host replication apparatus.
"Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex"
Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.
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phi29 DNA-protein p3 is replicated to full length with the DNA polymerase and the terminal protein as the only proteins, which is the reconstitution behind the IDA row for viral DNA genome replication and behind EC 2.7.7.7 in the UniProt entry.
"A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described."
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The same polymerase performs both the initiation chemistry and the elongation, so initiation and strand elongation are separately attributable to this one protein.
"the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA"
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ATP is required at neither step, ruling out a host primase or an ATP-dependent helicase in the minimal system.
"ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps."
Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.
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Processivity and strand displacement are intrinsic to the polymerase, requiring no accessory protein, and are what make the two-protein replication system sufficient.
"the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process"
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Chains greater than 70 kb are synthesised on primed M13 DNA, roughly four genome lengths, and template secondary structure does not impede the enzyme.
"the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs"
A conserved 3'->5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.
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Site-directed mutagenesis of the predicted exonuclease active site specifically inactivates the 3'-5' exonuclease of phi29 DNA polymerase while leaving polymerisation intact, establishing a separate N-terminal catalytic site. This is the experimental reference UniProt gives for EC 3.1.11.-.
"specifically inactivated the 3'----5' exonuclease activity of the enzyme"
DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.
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The polymerase catalyses covalent attachment of dAMP to the terminal protein even without DNA, with the same O-5'-deoxyadenylyl-L-serine linkage, the same Ser232 specificity and the same YCDTD motif requirement as genuine initiation. This is the cleanest demonstration that the deoxynucleotidylation of the terminal protein is an activity of this protein, separable from template-directed polymerisation.
"the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP"
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Without a director DNA the reaction loses base specificity, so the template's role at initiation is to stabilise the complementary nucleotide rather than to provide the chemistry.
"the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif"
Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.
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The synthetic and degradative activities have independent metal binding sites, consistent with the N-terminal exonuclease and C-terminal polymerisation domains.
"The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase"
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Protein-primed initiation specifically prefers Mn2+, through a large increase in dATP affinity; this is a metal-activation property in vitro rather than evidence of a second physiological cofactor, so no manganese-binding term is proposed.
"the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator"
Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.
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The downstream template passes through a tunnel too small for duplex DNA, which is the structural basis of coupling polymerisation to strand displacement.
"This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands."
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Processivity is explained by topological encirclement of both the downstream template and the upstream duplex, which is also the structural basis of the DNA binding annotation.
"explained by its topological encirclement of both the downstream template and the upstream duplex DNA"
The phi29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition.
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The terminal protein's priming domain occupies the same cleft in the polymerase that duplex DNA uses during elongation, so the growing product must displace it; this is the structural account of the initiation-to-elongation transition.
"a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation"
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The polymerase/terminal-protein heterodimer cannot accommodate upstream template DNA, which is why initiation is restricted to the genome ends - origin specificity arising from the enzyme's architecture rather than from a sequence-recognition activity.
"this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome"
Phage phi29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication.
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Initiation is a template-instructed addition of 5'-dAMP to the terminal protein, and the internal template nucleotide used is dictated by the terminal protein's priming domain, with sliding back recovering the terminal nucleotides.
"the DNA polymerase, in a template-instructed reaction, adds 5'-dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP"
Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.
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Four independently isolated temperature-sensitive mutants of gene 2 were cloned and sequenced, and in vivo their lesions affect only the initial steps of replication; the purified ts2(24) protein is thermosensitive in both protein-primed initiation and elongation. This establishes that temperature-sensitive mutant material for this gene exists, which is relevant to the CACAO row whose ECO refinement is temperature-sensitive mutant phenotypic evidence.
"The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported."
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The in vivo phenotype of the ts2 lesions is restricted to the initial steps of replication, independent mutant-based support for the GO:0006270 claim.
"in vivo, the mutations only affect the initial steps of the replication process"
ΓΈ29 DNA polymerase residue Lys383, invariant at motif B of DNA-dependent polymerases, is involved in dNTP binding.
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Mutating the invariant motif B lysine abolishes dNTP incorporation with either DNA or terminal protein as primer while leaving DNA affinity and the exonuclease unaffected, so the nucleotide site is distinct from the DNA site and from the exonuclease site, and it is a dNTP site rather than a nucleoside site.
"mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected"
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The conservative K383R substitution raises the Km for dNTPs 200-fold, a direct measurement of nucleotide, not nucleoside, binding by this enzyme.
"the Km for dNTPs being 200-fold higher than that of the wild-type enzyme"