polb encodes DNA polymerase beta, the small single-subunit X-family repair polymerase that performs the synthesis step of base-excision repair. It combines two activities in one chain: an 8-kDa N-terminal domain that removes the 5'-deoxyribose-phosphate left by AP endonuclease cleavage, acting as a lyase through a Schiff-base intermediate at the active-site nucleophile, and a 31-kDa polymerase domain that fills the resulting single-nucleotide gap, distributively rather than processively, with two magnesium ions in the active site. It can also cleave 3' of an intact abasic site as an AP lyase. The protein is found in the nucleus, where repair occurs, and in the cytoplasm under normal conditions. The purified zebrafish enzyme has been characterised biochemically and behaves like its mammalian counterparts, with an alkaline pH optimum, a 30 degrees C temperature optimum and markedly higher catalytic efficiency with Mn2+ than Mg2+.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Nucleus (IBA, is_active_in). UniProt places pol beta in the nucleus and in the cytoplasm, and nuclear chromatin is where base-excision repair substrates arise. Reason: The compartment in which the annotated activities act on their substrate: genomic DNA. No zebrafish-specific localisation data exist, but the enzyme has no organellar targeting sequence and the family's repair role is nuclear. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. |
| GO:0006284 base-excision repair | IBA GO_REF:0000033 | ACCEPT | Summary: Base-excision repair (IBA from PTN000138687). Pol beta performs two of the pathway's steps itself: 5'-dRP excision and single-nucleotide gap filling [file UniProt FUNCTION], and the human ortholog carries this term by IDA and IMP. Reason: Participation is direct and catalytic rather than inferred from a requirement, and this is the process for which the enzyme's two activities are jointly specialised. Core biological process. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0003887 DNA-directed DNA polymerase activity | IBA GO_REF:0000033 | ACCEPT | Summary: DNA-directed DNA polymerase activity (IBA from PTN000138687, the X-family polymerase node): template-directed addition of a deoxyribonucleotide to a primer 3' end. The zebrafish enzyme was purified and its polymerase kinetics measured directly [PMID:22018137]. Reason: Nothing to adjudicate: the propagated and automatic assignments agree with a direct assay of this protein, and the reaction is the one UniProt records (EC 2.7.7.7). Core molecular function; the gap-filling context is carried by the base-excision repair rows. Supporting Evidence: PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: DNA binding (InterPro mapping). Pol beta binds gapped DNA through the 8-kDa domain and the polymerase thumb; UniProt annotates a DNA-binding region, and the zebrafish-specific P63D substitution sits in the 8-kDa hairpin implicated in DNA binding [PMID:22018137]. Reason: Accurate and necessary for the enzyme to work, but it is a property subsumed by the polymerase and lyase activities, which specify what is done to the bound DNA. Not core on its own. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt DNA-binding PMID:22018137 This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0003887 DNA-directed DNA polymerase activity | IEA GO_REF:0000120 | ACCEPT | Summary: DNA-directed DNA polymerase activity (Rhea:22508-mapped IEA): template-directed addition of a deoxyribonucleotide to a primer 3' end. The zebrafish enzyme was purified and its polymerase kinetics measured directly [PMID:22018137]. Reason: Nothing to adjudicate: the propagated and automatic assignments agree with a direct assay of this protein, and the reaction is the one UniProt records (EC 2.7.7.7). Core molecular function; the gap-filling context is carried by the base-excision repair rows. Supporting Evidence: PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Nucleus (UniProt subcellular-location mapping). UniProt places pol beta in the nucleus and in the cytoplasm, and nuclear chromatin is where base-excision repair substrates arise. Reason: The compartment in which the annotated activities act on their substrate: genomic DNA. No zebrafish-specific localisation data exist, but the enzyme has no organellar targeting sequence and the family's repair role is nuclear. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Cytoplasm (UniProt subcellular-location mapping). UniProt notes the protein is cytoplasmic under normal conditions, relocating to the nucleus in response to damage. Reason: A faithful transfer of the UniProt record and probably right, but the repair activities are exercised on nuclear DNA; the cytoplasmic pool is a resting distribution rather than a site of catalysis, and there is no zebrafish imaging. Kept without making it a core location. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. |
| GO:0006259 DNA metabolic process | IEA GO_REF:0000117 | MODIFY | Summary: DNA metabolic process (ARBA rule). The broadest possible process statement for a DNA polymerase; correct, but it conveys nothing beyond what the catalytic terms already say. Reason: Changed from removal for the same reason as the DNA repair row: an over-general electronic assignment is not a false one. The informative replacement is base-excision repair, the pathway in which both of this enzyme's activities operate. Proposed replacements: base-excision repair Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- |
| GO:0006281 DNA repair | IEA GO_REF:0000002 | MODIFY | Summary: DNA repair, from the InterPro polymerase-beta signature. True but generic: the repair pathway this enzyme belongs to is base-excision repair, which it is annotated to by IBA and ISS. Reason: Previously marked for removal; that overstates the problem, since the assignment is correct, only too broad for an enzyme whose pathway is known. Replaced by the specific child rather than deleted. Proposed replacements: base-excision repair Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- PMID:22018137 Eukaryotic DNA polymerase Ξ² (pol Ξ²), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. |
| GO:0016779 nucleotidyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: Nucleotidyltransferase activity from the InterPro domain mapping: correct at the superfamily level but silent about the polymer and the template requirement. The measured activity of this protein is template-directed DNA synthesis [PMID:22018137]. Reason: A two-level-too-general parent; the specific terms are supported by direct assay of the zebrafish enzyme and by the UniProt reaction. Proposed replacements: DNA-directed DNA polymerase activity Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). |
| GO:0034061 DNA polymerase activity | IEA GO_REF:0000120 | MODIFY | Summary: DNA polymerase activity (IEA) is the parent of the template-directed term. Zebrafish pol beta is a template-dependent enzyme assayed on a primer-template [PMID:22018137], so the DNA-directed child is the supported statement. Reason: The generic term would also cover template-independent polymerases such as TdT, which this enzyme is not; the specific child GO:0003887 is already annotated here by IDA, IBA, ISS and IEA. Proposed replacements: DNA-directed DNA polymerase activity Supporting Evidence: PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0051575 5'-deoxyribose-5-phosphate lyase activity | IEA GO_REF:0000116 | ACCEPT | Summary: 5'-deoxyribose-5-phosphate lyase activity (Rhea-mapped IEA). The 8-kDa domain excises the 5'-dRP group left after AP endonuclease incision, through a Schiff base at the active-site nucleophile; UniProt annotates that catalytic residue in the zebrafish sequence. Reason: The second core catalytic activity of this enzyme and the one that makes it the dedicated BER polymerase: without dRP removal the nick cannot be ligated. The zebrafish protein retains the 8-kDa lyase domain, whose sequence the characterisation paper analysed explicitly. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt Nucleophile; Schiff-base intermediate with DNA; for 5'-dRP lyase activity PMID:22018137 This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. |
| GO:0071897 DNA biosynthetic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: DNA biosynthetic process. Pol beta does synthesise DNA, one nucleotide at a time into a repair gap [file UniProt FUNCTION], so the term is accurate; it simply does not distinguish repair synthesis from replicative synthesis. Reason: Changed from removal: the enzyme genuinely performs DNA synthesis, and removing the term would deny that. Kept non-core because the biologically meaningful framing is repair-gap filling, captured by base-excision repair, and pol beta contributes nothing to genome replication. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap file:DANRE/polb/polb-uniprot.txt Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases. file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate |
| GO:0140078 class I DNA-(apurinic or apyrimidinic site) endonuclease activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Class I DNA-(apurinic or apyrimidinic site) endonuclease activity, i.e. AP lyase: cleavage 3' of an intact abasic site by beta-elimination rather than hydrolysis. UniProt records this as a secondary capability of pol beta. Reason: Real but ancillary: the physiological BER route has AP endonuclease incise 5' of the abasic site and pol beta remove the resulting dRP, which is the GO:0051575 activity. The AP lyase reaction on an intact AP site is a lesser-used capability of the same 8-kDa domain, so it is retained without being made core. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt It is also able to cleave sugar-phosphate bonds 3' to an intact AP site, acting as an AP lyase. file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt Nucleophile; Schiff-base intermediate with DNA; for 5'-dRP lyase activity |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: Nucleus (ISS from human POLB). UniProt places pol beta in the nucleus and in the cytoplasm, and nuclear chromatin is where base-excision repair substrates arise. Reason: The compartment in which the annotated activities act on their substrate: genomic DNA. No zebrafish-specific localisation data exist, but the enzyme has no organellar targeting sequence and the family's repair role is nuclear. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Cytoplasm (ISS from human POLB). UniProt notes the protein is cytoplasmic under normal conditions, relocating to the nucleus in response to damage. Reason: A faithful transfer of the UniProt record and probably right, but the repair activities are exercised on nuclear DNA; the cytoplasmic pool is a resting distribution rather than a site of catalysis, and there is no zebrafish imaging. Kept without making it a core location. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. |
| GO:0003887 DNA-directed DNA polymerase activity | IDA PMID:22018137 Characterization of DNA polymerase Ξ² from Danio rerio by ove... | ACCEPT | Summary: DNA-directed DNA polymerase activity, IDA on the purified zebrafish protein: recombinant Danio pol beta was expressed, purified and assayed by primer extension, with Km, kcat and kcat/Km determined for dTTP incorporation under Mg2+ and Mn2+ [PMID:22018137]. Reason: Direct measurement of the template-directed polymerase reaction on this gene product, the strongest evidence in the set, and the anchor for the IBA, ISS and automatic polymerase rows. Core molecular function. Supporting Evidence: PMID:22018137 We have succeeded in overproducing, purifying, and characterizing Danio pol Ξ². PMID:22018137 We investigated the kinetic constants Km and kcat, and the catalytic efficiency kcat/Km for Danio pol Ξ² in the presence of either Mg2+ or Mn2+. PMID:22018137 Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate |
| GO:0003887 DNA-directed DNA polymerase activity | ISS GO_REF:0000024 | ACCEPT | Summary: DNA-directed DNA polymerase activity (ISS from human POLB, P06746): template-directed addition of a deoxyribonucleotide to a primer 3' end. The zebrafish enzyme was purified and its polymerase kinetics measured directly [PMID:22018137]. Reason: Nothing to adjudicate: the propagated and automatic assignments agree with a direct assay of this protein, and the reaction is the one UniProt records (EC 2.7.7.7). Core molecular function; the gap-filling context is carried by the base-excision repair rows. Supporting Evidence: PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Cytoplasm (ISS from human POLB). UniProt notes the protein is cytoplasmic under normal conditions, relocating to the nucleus in response to damage. Reason: A faithful transfer of the UniProt record and probably right, but the repair activities are exercised on nuclear DNA; the cytoplasmic pool is a resting distribution rather than a site of catalysis, and there is no zebrafish imaging. Kept without making it a core location. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. file:DANRE/polb/polb-uniprot.txt SUBCELLULAR LOCATION: Nucleus file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. |
| GO:0006284 base-excision repair | ISS GO_REF:0000024 | ACCEPT | Summary: Base-excision repair (ISS from human POLB). Pol beta performs two of the pathway's steps itself: 5'-dRP excision and single-nucleotide gap filling [file UniProt FUNCTION], and the human ortholog carries this term by IDA and IMP. Reason: Participation is direct and catalytic rather than inferred from a requirement, and this is the process for which the enzyme's two activities are jointly specialised. Core biological process. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0006303 double-strand break repair via nonhomologous end joining | IBA GO_REF:0000033 | REMOVE | Summary: Double-strand break repair via nonhomologous end joining (IBA from PTN000138687, the X-family polymerase node, with yeast Pol4, S. pombe, Arabidopsis and human POLL as donors). NHEJ gap filling in vertebrates is done by DNA polymerases lambda and mu, which are recruited to the Ku-XRCC4-ligase IV synapse through N-terminal BRCT domains that pol beta does not have; pol beta is a 336-residue protein consisting only of the 8-kDa lyase domain and the polymerase domain [file UniProt; PMID:22018137]. Reason: The node placement spreads an activity of one branch of the X-family across the whole family. The experimental grounding for NHEJ in this family sits on POLL (IDA and IMP) and POLM, not on pol beta: human POLB itself holds GO:0006303 only by the same IBA, so there is no experimental descendant anywhere on the pol beta branch. The structural reason is concrete - the BRCT module that tethers pol lambda and pol mu to the end-joining machinery is absent from pol beta, whose domain content the zebrafish characterisation paper describes directly - and the enzyme's substrate is a preincised single-nucleotide gap, not a synapsed double-strand break. Removing the term does not deny that pol beta can extend a primer in vitro on many substrates. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000138687 SOURCE BAD The ancestral node covers all X-family polymerases; NHEJ participation is a derived property of the pol lambda/pol mu/Pol4 branch, which acquired BRCT-mediated recruitment to the end-joining complex, so placing it at the family node carries it onto the pol beta branch incorrectly. UniProtKB:Q9UGP5 SUPPORTS SOURCE BUT NOT TARGET Human POLL legitimately carries NHEJ by IDA and IMP, but it is a BRCT-containing paralog, not the ortholog of this gene. SGD:S000000607 SUPPORTS SOURCE BUT NOT TARGET Yeast POL4 is the budding-yeast NHEJ polymerase and is likewise a BRCT-containing member; budding yeast has no pol beta orthologue to separate the two roles. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Belongs to the DNA polymerase type-X family. PMID:22018137 This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0006974 DNA damage response | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: DNA damage response (ISS from human POLB), a broad process term covering cellular reactions to genomic insult. For this enzyme the response consists of the repair synthesis it catalyses; the human ortholog also carries the term by IMP. Reason: Correct but at a level that says less than the base-excision repair rows, which name the actual pathway and the steps the protein performs. Retained as context. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt Repair polymerase that plays a key role in base-excision repair. file:DANRE/polb/polb-uniprot.txt Note=Cytoplasmic in normal conditions. PMID:22018137 Eukaryotic DNA polymerase Ξ² (pol Ξ²), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. |
| GO:0016829 lyase activity | ISS GO_REF:0000024 | MODIFY | Summary: Lyase activity (ISS from human POLB) is the root-level parent of the two lyase reactions this enzyme performs: 5'-dRP removal and AP-site beta-elimination. Reason: Uninformative at this level; the specific lyase terms are annotated and supported. Replaced rather than removed because the lyase chemistry itself is genuine and structurally localised to the 8-kDa domain. Proposed replacements: 5'-deoxyribose-5-phosphate lyase activity class I DNA-(apurinic or apyrimidinic site) endonuclease activity Supporting Evidence: file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt It is also able to cleave sugar-phosphate bonds 3' to an intact AP site, acting as an AP lyase. file:DANRE/polb/polb-uniprot.txt Nucleophile; Schiff-base intermediate with DNA; for 5'-dRP lyase activity |
| GO:0034061 DNA polymerase activity | ISS GO_REF:0000024 | MODIFY | Summary: DNA polymerase activity (ISS from human POLB) is the parent of the template-directed term. Zebrafish pol beta is a template-dependent enzyme assayed on a primer-template [PMID:22018137], so the DNA-directed child is the supported statement. Reason: The generic term would also cover template-independent polymerases such as TdT, which this enzyme is not; the specific child GO:0003887 is already annotated here by IDA, IBA, ISS and IEA. Proposed replacements: DNA-directed DNA polymerase activity Supporting Evidence: PMID:22018137 We performed extensive biochemical characterization of Danio pol Ξ² in comparison with rat pol Ξ², which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30Β°C). file:DANRE/polb/polb-uniprot.txt Reaction=DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate file:DANRE/polb/polb-uniprot.txt through its DNA polymerase activity, it adds one nucleotide to the 3' end of the arising single-nucleotide gap |
| GO:0051575 5'-deoxyribose-5-phosphate lyase activity | ISS GO_REF:0000024 | ACCEPT | Summary: 5'-deoxyribose-5-phosphate lyase activity (ISS from human POLB). The 8-kDa domain excises the 5'-dRP group left after AP endonuclease incision, through a Schiff base at the active-site nucleophile; UniProt annotates that catalytic residue in the zebrafish sequence. Reason: The second core catalytic activity of this enzyme and the one that makes it the dedicated BER polymerase: without dRP removal the nick cannot be ligated. The zebrafish protein retains the 8-kDa lyase domain, whose sequence the characterisation paper analysed explicitly. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt Nucleophile; Schiff-base intermediate with DNA; for 5'-dRP lyase activity PMID:22018137 This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. |
| GO:0140078 class I DNA-(apurinic or apyrimidinic site) endonuclease activity | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Class I DNA-(apurinic or apyrimidinic site) endonuclease activity, i.e. AP lyase: cleavage 3' of an intact abasic site by beta-elimination rather than hydrolysis. UniProt records this as a secondary capability of pol beta. Reason: Real but ancillary: the physiological BER route has AP endonuclease incise 5' of the abasic site and pol beta remove the resulting dRP, which is the GO:0051575 activity. The AP lyase reaction on an intact AP site is a lesser-used capability of the same 8-kDa domain, so it is retained without being made core. Supporting Evidence: file:DANRE/polb/polb-uniprot.txt It is also able to cleave sugar-phosphate bonds 3' to an intact AP site, acting as an AP lyase. file:DANRE/polb/polb-uniprot.txt the DNA backbone is nicked at the abasic site by an apurinic/apyrimidic (AP) endonuclease, and POLB removes 5'-deoxyribose- file:DANRE/polb/polb-uniprot.txt Nucleophile; Schiff-base intermediate with DNA; for 5'-dRP lyase activity |
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Download this section (compressed HTML)Q: Does zebrafish pol beta have the 5'-dRP lyase activity that defines the mammalian enzyme, given that the only biochemical study of the fish protein assayed polymerase activity alone?
Q: Is the cytoplasmic localisation recorded for pol beta a resting distribution that shifts to the nucleus on DNA damage in zebrafish, as reported for the mammalian protein?
Experiment: Assay purified zebrafish pol beta on a preincised AP-site substrate for dRP release and trap the Schiff-base intermediate by borohydride crosslinking, comparing wild type with the active-site lysine mutant.
Hypothesis: Zebrafish pol beta removes 5'-dRP through the conserved Schiff-base nucleophile.
Type: biochemical
Experiment: Generate a polb null line and compare survival and lesion repair after methyl methanesulfonate or hydrogen peroxide versus ionising radiation, testing the base-excision-specific role against the removed nonhomologous end-joining annotation.
Hypothesis: polb loss sensitises zebrafish to alkylating and oxidative base damage but not to double-strand breaks.
Type: genetic sensitivity assay
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