APN1 (YKL114C) encodes the major apurinic/apyrimidinic (AP) endonuclease and DNA 3'-repair diesterase of Saccharomyces cerevisiae, the eukaryotic counterpart of Escherichia coli endonuclease IV (Nfo). It is a 367-residue monomeric metalloenzyme of the AP endonuclease 2 (endonuclease IV) family, built on a divalent-metal-dependent TIM-barrel fold whose active-site pocket coordinates a trinuclear zinc cluster; two of the coordinating residues, Glu158 and Asp192, are each individually essential for activity in vivo. Apn1 incises the phosphodiester backbone 5' of abasic sites created by DNA glycosylases or by spontaneous base loss, generating the 3'-hydroxyl primer that DNA polymerase and ligase need, and it removes 3'-blocking deoxyribose fragments such as 3'-phosphoglycolaldehyde and 3'-phosphoglycolate from oxidative single-strand breaks. Gene disruption shows it supplies at least 97% of both activities in yeast extracts, and in extracts lacking it the 5'-incision step of base excision repair on uracil-containing DNA is undetectable; a second, minor AP endonuclease (Apn2) is partly redundant with it. The same active site supports a robust progressive 3'-to-5' exonuclease activity on duplex but not single-stranded DNA, which excises 3'-terminal mismatches, 3'-blocking groups and misincorporated 8-oxoguanine and constitutes a glycosylase-independent route for removing 8-oxoG from newly synthesised DNA. Purified Apn1 also hydrolyses 3'-phosphotyrosyl links of the kind formed by trapped topoisomerase I, although epistasis analysis indicates this activity is incidental to topoisomerase-lesion repair in vivo. Apn1 is concentrated in the nucleus by a bipartite basic nuclear localisation signal in its C-terminal extension, and truncations that fail to concentrate there also fail to protect cells against oxidative and alkylating agents; a minor pool reported to depend on Pir1p reaches the mitochondria, where loss of Apn1 raises the mitochondrial mutation rate. Loss of APN1 confers hypersensitivity to alkylating and oxidising agents and a spontaneous mutator phenotype that is strongly synergistic with loss of the 8-oxoguanine glycosylase Ogg1.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from IPR001719 (AP endonuclease family 2). Apn1 does bind DNA - the Glu158Gly active-site variant is catalytically crippled yet retains DNA binding - but the term is a substrate-binding capability that supports the catalytic functions rather than a function in its own right. Reason: True of the gene and as precise as the available evidence allows. The binding assay in the structure-function paper is not visible in the abstract-only cached record, so a refinement to GO:0003684 damaged DNA binding cannot be justified from what can be read; the generic term is kept, but as a supporting capability, not a core function (the core functions are the catalytic ones). Supporting Evidence: PMID:12767225 the Apn1 Glu158Gly mutant, but not the E. coli endonuclease IV Glu145Gly mutant, is able to bind DNA. |
| GO:0003906 DNA-(apurinic or apyrimidinic site) endonuclease activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA at PTN000485019 (the endonuclease IV / AP endonuclease 2 family node), seeded by E. coli Nfo (P0A6C1), C. elegans apn-1 and APN1 itself. The node placement is sound - AP-site incision is the defining activity of the family, and APN1 is the characterised eukaryotic member, with its own IDA and IMP rows for the same term. Reason: The target is squarely inside the clade that inherited the activity, and its own experimental annotations are among the descendant evidences behind the IBD - which is expected and is a marker that the claim is experimentally grounded on the target itself, not circular support. Supporting Evidence: PMID:1693433 The DNA and predicted amino acid sequences for the APN1 gene are homologous to those for the nfo gene encoding DNA endonuclease IV of Escherichia coli. PMID:1693433 Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. |
| GO:0003906 DNA-(apurinic or apyrimidinic site) endonuclease activity | IDA PMID:12767225 Characterization of two independent amino acid substitutions... | ACCEPT | Summary: CACAO row (also made independently by SGD from the same paper). Jilani et al. 2003 is a structure-function analysis of Apn1's abasic-site cleavage: glycine substitutions at the two zinc-coordinating active-site residues Glu158 and Asp192 abolish the enzyme's biological function, and Asp192Gly is completely inactive. The CACAO term matches what the paper is about. Reason: CACAO assessment: the term is the right term at the right granularity for this paper, on the right protein, with an evidence code that fits an in vitro activity assay on purified mutant proteins. The cached record is abstract-only and the GONUTS page for PMID:12767225 is offline, so the specific figure cannot be cited; but the abstract itself states the activity measurement, and SGD curated the same term from the same paper, so there is nothing to second-guess. Supporting Evidence: PMID:12767225 Moreover, Apn1 Asp192Gly completely lacks enzymatic activity, while the activity of the E. coli counterpart Asp179Gly is reduced by approximately 40-fold. PMID:12767225 We now report the first structure-function analysis of the eukaryotic counterpart of endonuclease IV, yeast Apn1. |
| GO:0003906 DNA-(apurinic or apyrimidinic site) endonuclease activity | IMP PMID:1693433 Yeast structural gene (APN1) for the major apurinic endonucl... | ACCEPT | Summary: IMP from the cloning and gene-disruption paper, which showed that disrupting APN1 removes at least 97% of the AP endonuclease activity of yeast extracts. This is the foundational evidence that APN1 encodes the major AP endonuclease. Supporting Evidence: PMID:1693433 Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: IBA at PTN000485021 with APN1 and C. elegans apn-1 among the descendant evidences. Nuclear action is where the enzyme's AP-site incision is required, as shown by the NLS-truncation series. Reason: The node placement is consistent with the target's own immunofluorescence evidence, and the functional test is explicit - derivatives that fail to concentrate in the nucleus fail to complement an apn1 deletion for damage resistance, so this is where the activity matters. Supporting Evidence: PMID:7690756 Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain. |
| GO:0005634 nucleus | IDA PMID:12767225 Characterization of two independent amino acid substitutions... | ACCEPT | Summary: CACAO row from the Jilani 2003 structure-function paper. The cached record is abstract-only and the abstract does not mention localisation at all, so what the student saw cannot be checked; the GONUTS page for this PMID is offline (the site returns a maintenance notice) and the Internet Archive is unreachable from this container. The claim itself is correct and independently established by IDA in PMID:7690756. Reason: CACAO assessment: right protein, right compartment, plausible for the full text (a structure-function study would normally check that the inactive variants are still nuclear), but the supporting figure is not verifiable from the cached abstract. Curator deference applies - an experimental annotation is not overturned because the abstract is silent - and the location is independently IDA-supported, so the row is accepted on the strength of PMID:7690756 rather than on anything quotable from PMID:12767225. The pattern worth noting for CACAO is one paper being annotated with the protein's whole known repertoire rather than only what that paper shows. Supporting Evidence: PMID:7690756 We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. |
| GO:0005634 nucleus | IDA PMID:7690756 Intracellular localization of the Apn1 DNA repair enzyme of ... | ACCEPT | Summary: IDA by indirect immunofluorescence, with a C-terminal deletion series mapping two basic lysine/arginine clusters as the nuclear transport signal. The primary and functionally relevant location of Apn1. Supporting Evidence: PMID:7690756 We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. PMID:7690756 The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the UniProt subcellular location (Nucleus) to GO:0005634. The curated UniProt statement rests on the same immunofluorescence evidence and agrees with the IDA and IBA rows. Supporting Evidence: file:yeast/APN1/APN1-uniprot.txt SUBCELLULAR LOCATION: Nucleus. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA at PTN000485021 propagating a mitochondrial location for the family. For APN1 the descendant evidence is its own IDA from PMID:11238901, and an AP endonuclease acting on the mitochondrial genome is a coherent claim, supported functionally by the elevated mitochondrial mutation rate of apn1 mutants. Reason: Root-organelle row examined as the policy requires: the synthesis rests on a single laboratory's localisation result in one organism, which UniProt has not adopted (the flat file records only the nucleus). The organelle claim is retained because the functional genetics support it, but it is peripheral - the nucleus is where the enzyme is concentrated and where its activity is shown to be needed - so it is kept as non-core rather than accepted as a core location, and no sub-compartment term is added, there being only one line of evidence and no submitochondrial assay. Supporting Evidence: PMID:11238901 Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. |
| GO:0005739 mitochondrion | IDA PMID:11238901 Pir1p mediates translocation of the yeast Apn1p endonuclease... | KEEP AS NON CORE | Summary: IDA from Vongsamphanh et al. 2001, which reported a mitochondrial pool of Apn1p whose import depends on Pir1p competing with the nuclear transport machinery, and showed that apn1 and pir1 mutants exposed to MMS accumulate mitochondrial mutations. Reason: Root-organelle row with direct evidence from an assay that cannot resolve a sub-compartment, so the organelle term is the right granularity and is not modified. It is kept as non-core because it is a minor pool from a single laboratory that UniProt has not taken up, and the enzyme's characterised role is nuclear. The accompanying mutation-rate data are a phenotype of losing the protein, not evidence for a separate mitochondrial process term. Supporting Evidence: PMID:11238901 Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. PMID:11238901 pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. |
| GO:0006281 DNA repair | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from IPR001719. Broader than the base-excision repair rows, but it says something they do not - Apn1 also acts outside canonical BER, cleaning 3'-blocking groups from oxidative single-strand breaks and excising 3'-terminal lesions from newly synthesised DNA. Reason: A pathway-level process term beside a specific one is informative rather than redundant here, because the gene's repair activities are not all within base-excision repair. Not marked TOO_GENERAL for that reason. Supporting Evidence: PMID:1693433 DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. PMID:16024777 Results obtained with the cell extracts support the idea that in vivo Apn1 exonuclease might be involved in the repair of single-strand breaks containing 3β²-blocking groups and mismatched nucleotides. |
| GO:0006284 base-excision repair | IBA GO_REF:0000033 | ACCEPT | Summary: IBA at the same family node as the AP endonuclease activity. Apn1 performs the AP-site incision step of BER itself, so this is participation and not merely a requirement. Reason: The node placement is sound and the target has its own IDA for the term; the family's defining activity is the incision step of base-excision repair. Supporting Evidence: PMID:9295360 we demonstrate that yeast BER requires Apn1 protein, an Escherichia coli endonuclease IV homolog |
| GO:0006284 base-excision repair | IDA PMID:9295360 Molecular mechanism of base excision repair of uracil-contai... | ACCEPT | Summary: IDA from a reconstituted yeast cell-free BER system on uracil-containing DNA, in which extracts of an apn1 deletion mutant show no detectable 5'-incision at AP sites. Apn1 executes a step of the pathway, so the process term is correctly applied. Supporting Evidence: PMID:9295360 In extracts of an apn1 deletion mutant, the 5'-incision at AP (apurinic/apyrimidinic) sites is not detectable, supporting the notion that yeast contains only one major 5'-AP endonuclease. |
| GO:0008081 phosphoric diester hydrolase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA propagating the family's 3'-repair diesterase activity, but onto a top-level esterase class rather than a term that says which phosphodiester is hydrolysed. The specific activity is removal of 3'-blocking deoxyribose fragments from DNA strand breaks. Reason: Correct but far more general than the evidence supports. GO:0090580 (phosphodiesterase activity, acting on 3'-phosphoglycolate-terminated DNA strands) is an is_a descendant of GO:0008081 and is the term human APEX1 carries for the same activity; the yeast enzyme was purified on a 3'-blocked-DNA substrate and is reported to hydrolyse a 3'-phosphoglycolate terminus. Both GO:0008081 rows are modified the same way. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000485019 SUPPORTS TRANSFER The node's judgement that the family hydrolyses 3'-blocked DNA termini is right for APN1; only the term chosen to express it is too broad. Proposed replacements: phosphodiesterase activity, acting on 3'-phosphoglycolate-terminated DNA strands Supporting Evidence: PMID:1693433 Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. PMID:12397185 Recently, it has been demonstrated that, like Apn1 and Apn2 ( 30 , 36 ), purified Tdp1 can hydrolyze a PG residue from the 3β² end of DNA ( 16 ). |
| GO:0008081 phosphoric diester hydrolase activity | IDA PMID:3056935 Yeast DNA diesterase for 3'-fragments of deoxyribose: purifi... | MODIFY | Summary: IDA from the purification of the yeast 3'-phosphoglycolaldehyde diesterase to homogeneity - the activity by which this protein was first isolated. The assay is specifically the hydrolytic removal of a 3'-blocking deoxyribose fragment from DNA, which the generic term does not convey. Reason: The underlying assay is a DNA 3'-end diesterase measurement, so GO:0090580 captures it while GO:0008081 places the enzyme among all phosphodiesterases. The same replacement is proposed for the IBA row on this term. Proposed replacements: phosphodiesterase activity, acting on 3'-phosphoglycolate-terminated DNA strands Supporting Evidence: PMID:3056935 We have used a synthetic DNA substrate containing 3'-phosphoglycolaldehyde esters to identify and purify to physical homogeneity the major yeast diesterase that removes such nucleotide fragments. PMID:3056935 The restoration of normal 3'-OH nucleotide primers is the essential first step in the excision repair of these radical-induced strand breaks. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the AP endonuclease family 2 signature and its zinc-binding site signature (IPR018246). The trinuclear zinc cluster is a characterised feature of this active site, and UniProt curates three Zn(2+) ions with nine binding-site features on P22936. Reason: Supported by curated UniProt cofactor and binding-site annotation and by the structure-function paper, which identifies the Apn1 residues corresponding to the zinc-coordinating Endo IV residues and shows that substituting them destroys activity. Zinc coordination is integral to the catalytic mechanism, not an incidental property. Supporting Evidence: file:yeast/APN1/APN1-uniprot.txt Note=Binds 3 Zn(2+) ions.; PMID:12767225 Both Glu145 and Asp179 are among nine amino acid residues within the active site pocket of endonuclease IV that coordinate the position of a trinuclear Zn cluster required for efficient phosphodiester bond cleavage. |
| GO:0008270 zinc ion binding | RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr... | ACCEPT | Summary: RCA from the yeast zinc-proteome survey, which assembled 582 known or potential zinc-binding proteins by combining domain and motif searches with proteomic quantification. For Apn1 the computational assignment coincides with the independently characterised trinuclear zinc site. Reason: A computational proteome-wide assignment on its own would be weak, but it agrees with curated UniProt cofactor annotation and with the active-site description in the structure-function literature, so the claim holds and the row reinforces the InterPro row. Supporting Evidence: PMID:30358795 The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. file:yeast/APN1/APN1-uniprot.txt Note=Binds 3 Zn(2+) ions.; |
| GO:0008296 3'-5'-DNA exonuclease activity | IDA PMID:12767225 Characterization of two independent amino acid substitutions... | MODIFY | Summary: CACAO row from Jilani 2003. The exonuclease activity is genuinely an Apn1 activity, but it was characterised elsewhere: Ishchenko et al. showed that purified Apn1 degrades duplex DNA and not single-stranded DNA, which makes GO:0008311 (double-stranded DNA 3'-5' DNA exonuclease activity) the term the data support. The cached Jilani record is abstract-only and its abstract does not mention an exonuclease assay, and the GONUTS page for the PMID is offline, so the student's figure cannot be checked. Reason: CACAO assessment: wrong granularity rather than a wrong claim. The parent term leaves the substrate unspecified, while the yeast enzyme is explicitly duplex-specific, and APN1 already carries the specific descendant GO:0008311 from PMID:16024777. Curator deference means the row is refined rather than removed - the activity is real, and this is an experimental annotation whose full text cannot be read here. Proposed replacements: double-stranded DNA 3'-5' DNA exonuclease activity Supporting Evidence: PMID:16024777 Using these substrates, we found that increasing concentrations of the purified Apn1 protein degraded the duplex DNAs (Fig. 1B , lanes 7 to 17) but not the single-stranded DNA (lanes 3 to 5). |
| GO:0008311 double-stranded DNA 3'-5' DNA exonuclease activity | IDA PMID:16024777 The 3'->5' exonuclease of Apn1 provides an alternative pathw... | ACCEPT | Summary: IDA from a dedicated biochemical characterisation. Purified Apn1 progressively degrades recessed, nicked and blunt duplex substrates but not single-stranded DNA, removes 3'-terminal mismatches, 8-oxoG and tetrahydrofuran abasic residues, and does so with efficiency comparable to its AP endonuclease activity. Substrate specificity and kinetics are both measured, so the duplex-specific term is exactly right. Reason: Directly demonstrated with purified protein and corroborated in extracts, where the activity is lost in apn1 deletion strains; genetically it provides an Ogg1-independent route for removing misincorporated 8-oxoG. A core activity of the protein alongside the endonuclease and diesterase functions. Supporting Evidence: PMID:16024777 Using these substrates, we found that increasing concentrations of the purified Apn1 protein degraded the duplex DNAs (Fig. 1B , lanes 7 to 17) but not the single-stranded DNA (lanes 3 to 5). PMID:16024777 In the case of Apn1, we demonstrate that it contains progressive exonuclease activity, which is as efficient as its AP endonuclease. |
| GO:0017005 3'-tyrosyl-DNA phosphodiesterase activity | IDA PMID:12397185 Repair of topoisomerase I covalent complexes in the absence ... | KEEP AS NON CORE | Summary: IDA from Fig. 3A of Liu et al. 2002: purified Apn1 removes a 3'-tyrosine efficiently from nicked and tailed duplex substrates, weakly from a blunt duplex and not at all from single-stranded DNA, and the corresponding activity in extracts is lost in apn1 cells. The activity is real, but the same paper's epistasis analysis shows it does not contribute to repair of topoisomerase I covalent complexes in vivo. Reason: The molecular function is directly demonstrated and the term is precise, so it is kept; but the authors explicitly conclude the esterase activity is incidental, and tdp1 is epistatic over apn1 apn2 for topoisomerase damage, so this should not be read as a core biological function of Apn1. Supporting Evidence: PMID:12397185 The purified enzyme removed tyrosine efficiently from the 3β² end of a synthetic oligonucleotide in the context of a nicked duplex or tailed duplex substrate. PMID:12397185 Thus, we infer that the esterase activity shown in Fig. 3 A is incidental, and it is some other activity of the AP endonucleases that works in the same pathway as does Tdp1. |
| GO:0017005 3'-tyrosyl-DNA phosphodiesterase activity | IDA PMID:12767225 Characterization of two independent amino acid substitutions... | KEEP AS NON CORE | Summary: CACAO row from Jilani 2003, whose abstract does not mention tyrosyl-DNA phosphodiesterase activity; the activity was demonstrated in PMID:12397185, from which SGD made the parallel row. The cached record is abstract-only and the GONUTS page for the PMID is offline, so the student's evidence statement could not be inspected. Reason: CACAO assessment: the claim is true of the protein but is not demonstrably what this paper shows - it reads as the same over-harvesting of one paper for the protein's whole activity repertoire seen in the other CACAO rows. Curator deference forbids removing an experimental annotation on the strength of a silent abstract, and the term matches a genuine in vitro activity, so the row is kept; it is non-core because the demonstrating paper's own genetics call the activity incidental in vivo. Supporting Evidence: PMID:12397185 The purified enzyme removed tyrosine efficiently from the 3β² end of a synthetic oligonucleotide in the context of a nicked duplex or tailed duplex substrate. PMID:12397185 Thus, we infer that the esterase activity shown in Fig. 3 A is incidental, and it is some other activity of the AP endonucleases that works in the same pathway as does Tdp1. |
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Download this section (compressed HTML)Q: Is the mitochondrial pool of Apn1 reproducible outside the originating laboratory, and does a Pir1-independent import route exist? UniProt still records only a nuclear location, and the claim rests on one 2001 study.
Q: Which activity of Apn1 - AP-site incision, 3'-diesterase or 3'-to-5' exonuclease - is rate-limiting for survival of each class of damaging agent, given that a single active site carries all three?
Q: Does the 3'-to-5' exonuclease act on DNA handed over by a stalled polymerase, as the proposed model requires, and if so which replication factor recruits it?
Experiment: Construct a panel of active-site and substrate-channel variants (beyond E158G and D192G), measure AP endonuclease, 3'-diesterase, 3'-to-5' exonuclease and tyrosyl-DNA phosphodiesterase activity for each purified protein, and score the matched strains for sensitivity to methyl methanesulfonate, bleomycin, hydrogen peroxide and camptothecin and for spontaneous mutation rate in an ogg1 background.
Hypothesis: The three activities of Apn1 are separable by active-site mutation, so their individual contributions to damage resistance can be assigned.
Experiment: Re-examine the localisation with a tagged Apn1 by submitochondrial fractionation with protease protection and by live imaging against an endoplasmic-reticulum marker to exclude mitochondria-associated membranes, then test whether Apn1 carrying an obligatory matrix targeting sequence and lacking the nuclear signal restores the mitochondrial mutation rate of apn1 cells.
Hypothesis: Apn1 reaches the mitochondrial matrix in a Pir1-dependent manner, and a mitochondrially restricted Apn1 is sufficient to suppress the mitochondrial mutator phenotype.
Experiment: Reconstitute primer extension with purified Pol delta, PCNA and RPA on 8-oxoG- and mismatch-terminated primers in the presence and absence of Apn1 and of exonuclease-selective Apn1 variants, and test for physical association by pull-down from yeast extracts under replication stress.
Hypothesis: The 3'-to-5' exonuclease of Apn1 operates on primer termini presented by a stalled replicative polymerase.
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