RAD27 (also known as RTH1 and YKL510; the Saccharomyces cerevisiae flap endonuclease 1) encodes the budding-yeast orthologue of FEN1, a magnesium-dependent structure-specific nuclease of the XPG/RAD2 family. Rad27 cleaves 5' single-stranded flaps, strongly preferring a double-flap substrate in which the upstream primer carries a one-nucleotide 3' tail; the tail positions cleavage exactly one nucleotide into the annealed region, so that every product is a nick that DNA ligase I (Cdc9) can seal. The same active site supports a processive 5'-to-3' exonuclease activity on duplex DNA and cleaves RNA flaps within RNA:DNA hybrid intermediates. Its central cellular role is Okazaki fragment maturation on the lagging strand, acting on the flaps produced by DNA polymerase delta strand-displacement synthesis together with PCNA (Pol30), which binds the Rad27 PIP box and both targets and stimulates the enzyme; a second, minor route uses Dna2 to trim long RPA-coated flaps before Rad27 makes the final cut, and DNA2 and RAD27 interact physically and are synthetically lethal. Rad27 also removes abasic-site flaps in long-patch base excision repair and trims incompatible 5' ends during nonhomologous end joining with Pol4 and Dnl4/Lif1; it can also cleave model R-loop 3' boundaries in vitro, albeit far less efficiently than canonical flaps. Loss of RAD27 leaves unligated Okazaki fragments and causes pronounced genome instability, including expansion and contraction of CAG trinucleotide repeats and severe ribosomal DNA copy-number changes, and the transcription-dependent DNA:RNA hybrids that accumulate after acute Rad27 depletion are best explained as a consequence of those lagging-strand discontinuities. Rad27 is predominantly nuclear and enriched in the nucleolus, and a functional pool is present in mitochondria, where it contributes to mitochondrial DNA integrity. Beyond replication, Rad27 is required for large loop repair, excising looped heteroduplexes from a 5' nick together with Pol delta, RFC and PCNA, and in ribonucleotide excision repair it removes the flap created after RNase H2 incises a ribonucleotide embedded in DNA.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006260 DNA replication | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic (PANTHER) inference that Rad27 acts in DNA replication. Correct but under-specified: its replication role is confined to lagging-strand Okazaki fragment maturation, where it cleaves the primer flap displaced by DNA polymerase delta so that Cdc9 can ligate the nick. Reason: Rad27 is not a general replication factor. Reconstituted yeast Okazaki fragment processing places it as the nuclease that removes the displaced flap, and rad27 mutants accumulate unligated Okazaki fragments in vivo. GO:0033567 states this precisely. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000118792 Β· FEN1 ancestral node SUPPORTS TRANSFER Node-level 'DNA replication' is correct for the family but understates a role confined to Okazaki fragment maturation. Proposed replacements: DNA replication, Okazaki fragment processing Supporting Evidence: PMID:16837458 A second pathway involves reiterative single nucleotide or short oligonucleotide displacement by pol delta and cleavage by FEN1. PMID:41140146 The rad27Ξ mutant accumulates Okazaki fragments in the rDNA region, without inducing the formation of detectable DSBs. |
| GO:0006281 DNA repair | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that Rad27 acts in DNA repair. Rad27 contributes to more than one repair route in budding yeast - long-patch base excision repair, end processing during nonhomologous end joining, and completion of double-strand-break-induced gene conversion - so the grouping term is appropriate. Reason: Unlike the replication annotation, which maps to a single pathway, the repair contributions genuinely span several sub-pathways, and the specific children (GO:0006284, GO:0006303) are separately annotated in this set. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN008960732 SUPPORTS TRANSFER Current PTHR11081 IBD for the broad repair clade containing RAD27/FEN1 and EXO1-related nucleases; the grouping term transfers to RAD27 because its yeast repair roles span BER, end joining, and recombinational repair contexts. Supporting Evidence: PMID:15342630 Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structure-specific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. |
| GO:0006310 DNA recombination | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Rad27 acts in DNA recombination. Rad27 loss does impair recombination-dependent processes such as MAT gene conversion, but through failure of the lagging-strand synthesis that accompanies repair synthesis, not through a dedicated recombination activity. Reason: Rad27 has no strand-exchange, resection or junction-resolution activity; its recombination phenotypes are attributable to its Okazaki-fragment role at the modified replication fork established after strand invasion. Retained as a genuine but non-core process. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN008960732 SUPPORTS TRANSFER Current PTHR11081 recombination IBD with RAD27 among the experimentally grounded descendants; the inherited annotation is defensible for repair synthesis after strand invasion, but secondary to the enzyme's Okazaki-fragment flap processing role. Supporting Evidence: PMID:10025407 We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that Rad27 is active in the nucleus. Nuclear localization of the yeast protein is independently recorded by the curated UniProt subcellular location and by the genome-wide GFP-fusion survey. Reason: Nuclear localization is where Rad27 encounters its nuclear replication and repair substrates, so the is_active_in qualifier is correct. No verbatim supporting quote is attached: the localization comes from dataset-level evidence (the GFP-fusion collection of PMID:14562095 and the UniProt curated location), not from a Rad27-specific sentence in any cached abstract. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000118792 SUPPORTS TRANSFER Current nuclear-localization IBD in PTHR11081; RAD27 is on the annotated lineage and has direct yeast localization support. |
| GO:0017108 5'-flap endonuclease activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of 5'-flap endonuclease activity. This is the defining catalytic activity of Rad27 and is demonstrated directly for the purified yeast enzyme. Reason: Core molecular function. Purified Rad27p cleaves 5' flap substrates, preferring a double-flap configuration and incising one nucleotide into the annealed region so that the product is directly ligatable. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000118612 SUPPORTS TRANSFER Current PTHR11081 IBD for FEN1-family 5'-flap endonuclease activity; RAD27 itself is among the direct experimental descendants. Supporting Evidence: PMID:11825897 We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. PMID:11825897 The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. |
| GO:0008409 5'-3' exonuclease activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of 5'-3' exonuclease activity. The yeast enzyme was purified as a 5'-3' exonuclease associated with DNA polymerase alpha before its identity as RTH1/RAD27 was established. Reason: Directly demonstrated for the purified yeast protein, which shows both processive 5'-3' exonucleolytic excision and flap endonuclease activity. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000118791 SUPPORTS TRANSFER Current eukaryote-level PTHR11081 IBD for the exonuclease subactivity, supported by direct RAD27 biochemistry as well as EXO1 and human FEN1 descendants. Supporting Evidence: PMID:9166764 We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. PMID:9166764 Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference that Rad27 is active in the cytoplasm. Rad27 acts on DNA, and the only extranuclear compartment in which it has a demonstrated substrate is the mitochondrion, which is part of the cytoplasm. Reason: 'is_active_in cytoplasm' implies a cytosolic site of action for which there is no substrate: Rad27's targets are nuclear and mitochondrial DNA. Direct evidence localizes Rad27p to the mitochondrial compartment, where its loss destabilizes mtDNA, so the mitochondrion is the specific extranuclear compartment in which it is active. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: COMPARTMENT OR COMPLEX MISMATCH GRANULARITY MISMATCH Sources checked: PANTHER:PTN000871783 SOURCE WEAK OR INFERRED Current fungal PTHR11081 IBD for a cytoplasm parent term, seeded only by yeast EXO1 and RAD27. RAD27 descends from and helped seed this node, but the target-specific extranuclear site of Rad27 action is the mitochondrial compartment, not a soluble cytosolic substrate. Proposed replacements: mitochondrion Supporting Evidence: PMID:19699691 Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. |
| GO:0000287 magnesium ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: Sequence-feature-based electronic transfer of magnesium ion binding. Mg2+ is the catalytic cofactor of the two-metal-ion FEN1/Rad27 active site. Reason: UniProt records that Rad27 binds two magnesium ions per subunit which probably participate in catalysis; the coordinating carboxylates are a conserved feature of the XPG/RAD2 nuclease fold. |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation of DNA binding. Rad27 must engage both the single-stranded flap and the flanking duplex, and its substrate recognition is exquisitely sensitive to the 3' tail of the upstream primer. Reason: General but correct and non-trivial for a structure-specific nuclease: Rad27 discriminates among branch-migration isomers of the same substrate, which requires specific DNA engagement. Supporting Evidence: PMID:11825897 FEN1 only cleaved those containing a 1-nucleotide 3'-tail. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to the root-level term 'catalytic activity'. True of Rad27 but conveys no information about what it does. Reason: GO:0003824 is a top-level grouping term. The same InterPro signatures support the specific 5'-flap endonuclease activity term, which is independently supported experimentally for the yeast enzyme. Proposed replacements: 5'-flap endonuclease activity Supporting Evidence: PMID:11825897 We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. |
| GO:0004518 nuclease activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping to nuclease activity, an appropriate domain-level statement for a protein carrying the flap endonuclease and XPG-I domains. Reason: A meaningful domain-derived assertion and a direct parent of the experimentally supported flap endonuclease and 5'-3' exonuclease activities. |
| GO:0005634 nucleus | IEA GO_REF:0000104 | ACCEPT | Summary: Electronic transfer of nuclear localization, confirmed directly for Rad27 by GFP tagging and immunofluorescence. Reason: Correct; the nucleus is the principal compartment of Rad27, corroborated by the genome-wide GFP survey and the curated UniProt location. |
| GO:0005654 nucleoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location vocabulary: nucleoplasm. Reason: Matches the curated UniProt subcellular location, which records Rad27 in nucleolus and nucleoplasm with relocalization to the nucleoplasm upon DNA damage. |
| GO:0005730 nucleolus | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location vocabulary: nucleolus. Consistent with Rad27's demonstrated role in maintaining rDNA repeat stability. Reason: UniProt records that Rad27 resides mostly in the nucleoli; independently, rad27 mutants accumulate Okazaki fragments specifically in the rDNA region and show severe rDNA copy-number instability. Supporting Evidence: PMID:41140146 Here, we demonstrate that Rad27/FEN1, a structure-specific nuclease in budding yeast, plays a crucial role in maintaining the stability of the ribosomal DNA (rDNA) repeats. |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation of mitochondrial localization, independently confirmed by direct evidence for a mitochondrial pool of Rad27p. Reason: Rad27p is present in the mitochondrial compartment and its loss compromises mtDNA integrity, so the electronic assignment is correct. Supporting Evidence: PMID:19699691 Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. |
| GO:0006281 DNA repair | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning annotation of DNA repair involvement, duplicating the IBA repair annotation. Reason: Correct grouping term; Rad27 participates in base excision repair, end joining and completion of recombinational repair. Supporting Evidence: PMID:15342630 Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structure-specific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. |
| GO:0006284 base-excision repair | IEA GO_REF:0000104 | ACCEPT | Summary: Electronic transfer of base-excision repair involvement. Rad27 removes the abasic-site-terminated flap generated by strand-displacement synthesis during long-patch BER. Reason: Core biological process, conserved with human FEN1 and supported by direct demonstration that yeast FEN1 cleaves RNA-containing flaps in the RNA:DNA hybrid intermediates of lagging-strand processing. The R-loop-specific BER experiments in the same study used the human enzyme. Supporting Evidence: PMID:36672839 We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. |
| GO:0008409 5'-3' exonuclease activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic transfer of 5'-3' exonuclease activity, duplicating the directly demonstrated activity of the purified yeast enzyme. Reason: Correct and independently supported by biochemistry on purified Rad27/RTH1. Supporting Evidence: PMID:9166764 We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. |
| GO:0016788 hydrolase activity, acting on ester bonds | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to 'hydrolase activity, acting on ester bonds'. True of any nuclease and therefore uninformative about Rad27. Reason: Too general to be useful; the same domain content supports at minimum nuclease activity and, given the flap endonuclease domain signature, the specific 5'-flap endonuclease term. Proposed replacements: 5'-flap endonuclease activity |
| GO:0017108 5'-flap endonuclease activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic transfer of 5'-flap endonuclease activity, duplicating the core experimentally supported molecular function. Reason: Correct and at the right level of specificity for Rad27. Supporting Evidence: PMID:11825897 We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. |
| GO:0043137 DNA replication, removal of RNA primer | IEA GO_REF:0000104 | ACCEPT | Summary: Electronic transfer of 'DNA replication, removal of RNA primer'. This is the precise replication role of Rad27, directly demonstrated in reconstituted yeast Okazaki fragment processing. Reason: Core biological process; reconstitution with purified S. cerevisiae proteins shows Rad27/FEN1 cleaving the flaps displaced by pol delta during primer removal. Supporting Evidence: PMID:16837458 Results showed that in the presence of PCNA and FEN1, pol delta displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length |
| GO:0005515 protein binding | IPI PMID:11805837 Systematic identification of protein complexes in Saccharomy... | REMOVE | Summary: IPI interaction with PCNA/Pol30 (P15873) detected in a proteome-wide mass-spectrometry survey of yeast protein complexes. Reason: The PCNA interaction is real, but this high-throughput row is recorded only as generic protein binding and has no more informative molecular-function replacement. The functional content - PCNA-dependent targeting and stimulation of Rad27 at the fork - is captured by the Okazaki fragment processing and flap endonuclease annotations. |
| GO:0005515 protein binding | IPI PMID:20967232 Subtle alterations in PCNA-partner interactions severely imp... | REMOVE | Summary: IPI interaction with PCNA/Pol30 (P15873) from a directed-evolution study of PCNA variants with altered partner affinities. Reason: The study is informative about the PCNA-Rad27 interface (tightening it is deleterious in vivo), but generic protein binding conveys no molecular function for Rad27, and GO has no specific PCNA-binding activity term to use as a replacement. Supporting Evidence: PMID:20967232 Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. |
| GO:0005515 protein binding | IPI PMID:7673186 Lagging strand DNA synthesis at the eukaryotic replication f... | REMOVE | Summary: IPI interaction with PCNA/Pol30 (P15873): PCNA binds FEN-1 and focuses it on flap and nicked substrates, stimulating activity 10-50-fold. Reason: Functionally central interaction, but the generic protein binding term carries no molecular-function information for Rad27 and has no precise PCNA-binding replacement. The consequence (targeted, stimulated flap cleavage during lagging-strand synthesis) is annotated via GO:0017108 and GO:0043137. Supporting Evidence: PMID:7673186 Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. PMID:7673186 Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold |
| GO:0005515 protein binding | IPI PMID:9121462 A yeast replicative helicase, Dna2 helicase, interacts with ... | REMOVE | Summary: IPI interaction with Dna2 (P38859): yFEN-1 coimmunopurifies with tagged Dna2, and the two genes interact genetically, including synthetic lethality of the double mutant. Reason: A biologically important partnership - Dna2 trims long RPA-coated flaps before Rad27 makes the final ligatable cut - but generic protein binding states nothing about Rad27's molecular function and has no evidence-backed replacement term here. The shared pathway is captured by the primer-removal and Okazaki fragment processing annotations. Supporting Evidence: PMID:9121462 both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2 PMID:9121462 dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal. |
| GO:0043570 maintenance of DNA repeat elements | IMP PMID:41140146 Rad27/FEN1 prevents accumulation of Okazaki fragments and ri... | ACCEPT | Summary: IMP of maintenance of DNA repeat elements: rad27 null mutants show severe ribosomal DNA copy-number instability and accumulate Okazaki fragments in the rDNA, independently of Fob1-mediated fork arrest or detectable double-strand breaks. Reason: Well-supported and mechanistically informative: it ties repeat instability directly to failed Okazaki fragment maturation rather than to break-induced processes, and the same phenotype arises in ligase-deficient cells while Exo1 and PCNA partially compensate for Rad27 loss. Supporting Evidence: PMID:41140146 Here, we demonstrate that Rad27/FEN1, a structure-specific nuclease in budding yeast, plays a crucial role in maintaining the stability of the ribosomal DNA (rDNA) repeats. PMID:41140146 Furthermore, Exonuclease 1 and PCNA partially compensate for the loss of Rad27 in rDNA stabilization. |
| GO:0005634 nucleus | IDA PMID:36672839 Flap Endonuclease 1 Endonucleolytically Processes RNA to Res... | ACCEPT | Summary: IDA that Rad27 is active in the nucleus, curated from the study of FEN1 cleavage of RNA:DNA hybrid intermediates. The yeast experiments in that paper are in vitro biochemistry; the cell-based localization data are from human fibroblasts. Reason: The nucleus is the correct compartment for Rad27's action on nuclear replication and BER intermediates, and the annotation is consistent with all other localization evidence. No supporting quote is attached because the yeast data in this paper are enzymatic rather than cell-biological; accepted in deference to the curator. |
| GO:0017108 5'-flap endonuclease activity | IDA PMID:36672839 Flap Endonuclease 1 Endonucleolytically Processes RNA to Res... | ACCEPT | Summary: IDA of 5'-flap endonuclease activity: yeast FEN1 efficiently cleaves RNA flaps in RNA:DNA hybrid intermediates generated during lagging-strand processing and BER. Reason: Core molecular function demonstrated directly on physiologically relevant flap substrates, and extended here to ribonucleotide-containing flaps. Supporting Evidence: PMID:36672839 We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. |
| GO:0043137 DNA replication, removal of RNA primer | IDA PMID:36672839 Flap Endonuclease 1 Endonucleolytically Processes RNA to Res... | ACCEPT | Summary: IDA of RNA primer removal during DNA replication, from assays on the RNA:DNA hybrid intermediates generated during lagging-strand processing. Reason: Core biological process; the assays specifically address the intermediates formed when the RNA primer of the downstream Okazaki fragment is displaced. Supporting Evidence: PMID:36672839 we initially examined the endo- and exonucleolytic activity of human and yeast FEN1 on a nicked RNA, an RNA flap formed within an RNA strand, and an RNA flap attached to a DNA strand PMID:36672839 The results indicate that both human and yeast FEN1 employed its endonucleolytic activity to cleave the RNA strands. |
| GO:0007534 gene conversion at mating-type locus | IMP PMID:10025407 Double-strand break repair in yeast requires both leading an... | KEEP AS NON CORE | Summary: IMP of gene conversion at the mating-type locus: rad27 deletion strongly inhibits completion of MAT switching after an HO-induced double-strand break, even in G1-arrested cells. Reason: The phenotype is genuine and the assay is specific, but mechanistically it reports the same lagging-strand requirement as the core Okazaki fragment role: the authors interpret strand invasion as creating a modified replication fork whose lagging-strand synthesis needs pol alpha, primase and Rad27. Retained as a real but non-core process rather than a distinct mating-type-specific function. Supporting Evidence: PMID:10025407 Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. |
| GO:0005634 nucleus | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: HDA of nuclear localization from the genome-wide GFP-fusion localization survey of budding yeast. Reason: High-throughput but high-quality systematic localization data, in agreement with all targeted evidence and with the curated UniProt subcellular location. The quote is omitted because the abstract describes the GFP-fusion collection rather than Rad27 specifically; the evidence is the dataset entry. |
| GO:0005829 cytosol | IDA PMID:22932476 The nuclear localization of SWI/SNF proteins is subjected to... | KEEP AS NON CORE | Summary: IDA of cytosolic localization from a screen for nuclear proteins that change compartment under hypoxia and reoxygenation. Reason: This records a condition-dependent redistribution observed in a survey of over 120 nuclear proteins whose localization changes under hypoxia, not a compartment in which Rad27 has a known substrate. It is plausible as an observation but is peripheral: Rad27's DNA substrates are nuclear and mitochondrial. Supporting Evidence: PMID:22932476 we have found that over 120 nuclear proteins with important functions ranging from transcriptional regulation to RNA processing exhibit altered cellular locations under hypoxia. |
| GO:0035753 maintenance of DNA trinucleotide repeats | IMP PMID:16079237 Interactions among DNA ligase I, the flap endonuclease and p... | ACCEPT | Summary: IMP of maintenance of DNA trinucleotide repeats: RAD27 mutations destabilize CAG repeat tracts more than most replication mutations, causing both expansions and contractions. Reason: Strong, specific genetic evidence, and mechanistically coherent with the flap endonuclease role - unremoved flaps equilibrate into hairpin-containing structures that seed repeat-length changes. Supporting Evidence: PMID:16079237 Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. |
| GO:0035753 maintenance of DNA trinucleotide repeats | IGI PMID:16079237 Interactions among DNA ligase I, the flap endonuclease and p... | ACCEPT | Summary: IGI of maintenance of DNA trinucleotide repeats: the PIP-box allele rad27-p combined with cdc9-p (DNA ligase I) or pol30-90 (PCNA) increases CAG expansion frequency more than additively. Reason: Genetic-interaction evidence that specifically implicates PCNA-mediated ordering of Rad27 and Cdc9 entry into Okazaki fragment joining, reinforcing rather than duplicating the IMP annotation. Supporting Evidence: PMID:16079237 Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. PMID:16079237 These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions. |
| GO:0005634 nucleus | IDA PMID:19699691 Evidence for a role of FEN1 in maintaining mitochondrial DNA... | ACCEPT | Summary: IDA of nuclear localization curated from the study that established a mitochondrial pool of Rad27p. The cached record for this paper is abstract-only and the abstract makes no statement about the nuclear compartment. Reason: Accepted in deference to the SGD curator, who read the full text; nuclear localization of Rad27 is in any case corroborated by the genome-wide GFP survey and the curated UniProt location. No supporting quote is attached because the only localization sentence in the cached abstract concerns the mitochondrion, which would not support this annotation. |
| GO:0005634 nucleus | IDA PMID:22932476 The nuclear localization of SWI/SNF proteins is subjected to... | ACCEPT | Summary: IDA of nuclear localization from the hypoxia relocalization study, in which the SGD curator scored Rad27 among the nuclear proteins surveyed under normoxic conditions. Reason: Agrees with the systematic GFP survey and with the targeted localization work; only the hypoxia-induced cytosolic scoring derived from the same study is peripheral. Supporting Evidence: PMID:22932476 we have found that over 120 nuclear proteins with important functions ranging from transcriptional regulation to RNA processing exhibit altered cellular locations under hypoxia |
| GO:0005739 mitochondrion | IDA PMID:19699691 Evidence for a role of FEN1 in maintaining mitochondrial DNA... | ACCEPT | Summary: IDA of mitochondrial localization: Rad27p is present in the mitochondrial compartment and rad27 deletion destabilizes mitochondrial DNA. Reason: Direct evidence for a functional mitochondrial pool, conserved with mammalian FEN1, and consistent with the demonstrated need for 5' flap removal during mitochondrial long-patch base excision repair. Supporting Evidence: PMID:19699691 Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. |
| GO:0006303 double-strand break repair via nonhomologous end joining | IDA PMID:15342630 Processing and joining of DNA ends coordinated by interactio... | KEEP AS NON CORE | Summary: IDA of double-strand break repair via nonhomologous end joining: FEN-1(Rad27) interacts physically and functionally with Pol4 and Dnl4/Lif1 and, together with them, processes and joins DNA ends with incompatible 5' termini. Reason: Well-supported biochemically, and Rad27's contribution is a specific one (trimming 5' ends to create ligatable termini) rather than a core identity. Retained as a real but secondary process, parallel to the flap-trimming role that Reactome assigns to human FEN1 in microhomology-mediated end joining. Supporting Evidence: PMID:15342630 we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5' ends. |
| GO:0008409 5'-3' exonuclease activity | IDA PMID:9166764 Purification and characterization of the DNA polymerase alph... | ACCEPT | Summary: IDA of 5'-3' exonuclease activity for the purified RTH1/Rad27 protein, which also displays strong flap endonuclease activity. Reason: Core molecular function, demonstrated on the purified yeast enzyme with mechanistic characterization of its exonucleolytic excision. Supporting Evidence: PMID:9166764 The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. |
| GO:0017108 5'-flap endonuclease activity | IDA PMID:11825897 Cleavage specificity of Saccharomyces cerevisiae flap endonu... | ACCEPT | Summary: IDA of 5'-flap endonuclease activity establishing that Rad27p prefers a double-flap substrate with a one-nucleotide 3' tail on the upstream primer and cleaves one nucleotide into the annealed region. Reason: The most precise biochemical definition of the Rad27 reaction: the cleavage position is set by the 3' tail and yields exclusively ligatable nicks, explaining why Rad27 and DNA ligase I must act in an ordered fashion. Supporting Evidence: PMID:11825897 Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. PMID:11825897 The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. |
| GO:0043137 DNA replication, removal of RNA primer | IDA PMID:16837458 Reconstituted Okazaki fragment processing indicates two path... | ACCEPT | Summary: IDA of RNA primer removal, from Okazaki fragment processing reconstituted with purified S. cerevisiae proteins, showing that FEN1 cleavage of pol delta-displaced flaps accounts for the bulk of primer removal. Reason: Core biological process demonstrated in a fully reconstituted system, and it also defines the division of labour with the Dna2-dependent long-flap route. Supporting Evidence: PMID:16837458 Results showed that in the presence of PCNA and FEN1, pol delta displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length PMID:16837458 One proposed pathway for flap removal involves pol delta displacement of long flaps, coating of those flaps by replication protein A (RPA), and sequential cleavage of the flap by Dna2 nuclease followed by flap endonuclease 1 (FEN1). |
| GO:0004523 RNA-DNA hybrid ribonuclease activity | IDA PMID:36672839 Flap Endonuclease 1 Endonucleolytically Processes RNA to Res... | NEW | Summary: Yeast FEN1 (Rad27) efficiently cleaves nicked RNA and RNA flaps within RNA:DNA hybrid intermediates using its endonuclease activity (Figures 1D-F and 2D-F of PMID:36672839, assayed on recombinant S. cerevisiae Rad27). PMID:40064914 also detects weak cleavage of synthetic R-loop 3' boundaries, without establishing that the RNA strand is cleaved or that this activity explains the in vivo hybrid phenotype. RAD27 currently carries no RNase H-type activity annotation, although its human ortholog does. Reason: This activity is what allows Rad27 to remove ribonucleotide-containing Okazaki primers directly. It was assayed on the purified yeast enzyme side by side with human FEN1, so IDA is the correct evidence code here. It is a conserved property of the family - human FEN1/DNase IV was shown to possess RNase H activity when first purified. Supporting Evidence: PMID:36672839 We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. PMID:36672839 The results indicate that both human and yeast FEN1 employed its endonucleolytic activity to cleave the RNA strands. PMID:40064914 In contrast, while the cleavage of R-loop 3β² boundaries was detectable (Fig.Β 3b, right panels), it was several orders of magnitude less efficient than for flaps (Fig.Β 3d, e). |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Rad27 performs the flap-excision step of ribonucleotide excision repair in a reconstitution with purified yeast enzymes (PMID:22864116), but GOA has no GO:1990516 ribonucleotide excision repair row for RAD27 (it carries the broader GO:0006281 DNA repair). Should SGD annotate RAD27 to GO:1990516 by IDA?
Q: The IBA 'is_active_in cytoplasm' annotation has no identifiable cytosolic substrate for Rad27. Should the PANTHER family annotation be re-scoped to the mitochondrion, the only extranuclear compartment where Rad27 has a demonstrated DNA substrate?
Q: How much of the rad27 delta genome-instability phenotype is attributable to unremoved flaps versus to the secondary recombination and checkpoint responses they trigger?
Q: Exo1 and PCNA partially compensate for loss of Rad27 in rDNA stabilization. Which step of Okazaki fragment maturation does Exo1 actually substitute for, and is the compensation nucleolytic or structural?
Q: In which quiescent, telomeric or DNA-damage contexts does the weak direct R-loop cleavage activity of Rad27 become physiologically relevant rather than secondary to flap accumulation?
Q: Does the cytosolic Rad27 signal reported under hypoxia represent regulated relocalization with a functional consequence, or redistribution of a nuclear pool with no substrate outside the nucleus and mitochondria?
Experiment: Okazaki fragment sequencing (OK-seq or equivalent) in rad27 delta, rad27-p (PIP-box) and nuclease-dead RAD27 strains, to separate the contributions of PCNA targeting and of catalysis to fragment maturation genome-wide and at the rDNA specifically.
Experiment: Reconstitution of yeast Okazaki fragment processing with defined ratios of Rad27, Dna2, RPA, Pol30 and Cdc9 to quantify the flux through the short-flap versus long-flap routes under near-physiological conditions.
Experiment: Mitochondrially targeted versus nuclear-restricted RAD27 alleles, scored for mtDNA point mutation and deletion rates, to test whether the mitochondrial pool is functionally required or incidental.
Experiment: Combine flap-competent, R-loop-cleavage-defective rad27 alleles with locus-specific DRIP in quiescent, telomeric and base-damage contexts to test whether Rad27's weak direct R-loop cleavage activity is ever the limiting activity in vivo.
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Download this section (compressed HTML)5' flap endonuclease, 5' to 3' exonuclease and gap endonuclease; required for Okazaki fragment processing and maturation, for long-patch base-excision repair and large loop repair (LLR), ribonucleotide excision repair; cleaves off RNA flap in a RNA:DNA hybrid intermediate during processing of DNA lagging strand; relocalizes to the cytosol in response to hypoxia; member of the S. pombe RAD2/FEN1 family
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