Gen (DmGEN, mus324) encodes the Drosophila ortholog of human GEN1 and yeast Yen1, a member of the XPG/RAD2 family of Mg2+-dependent structure-selective endonucleases (GEN/class IV subfamily) with closely spaced XPG-N and XPG-I nuclease domains, an H3TH DNA-binding region and a chromodomain-like region. Purified Gen cleaves 5' flaps (preferring blocked flaps), the lagging strand of replication-fork-like structures, splayed arms, and Holliday junctions, which it cuts symmetrically to give ligatable nicked duplexes after dimerizing on the junction; it also has a weak nick-dependent 5'-3' exonuclease activity. In flies Gen is the principal structure-selective nuclease for DNA damage tolerance: mutants are strongly hypersensitive to alkylating, crosslinking and replication-blocking agents and to ionizing radiation, with MUS81-MMS4 acting as a backup, and Gen becomes essential in the absence of the BLM or FANCM helicases. In interphase cells the protein appears largely cytoplasmic, as reported for its human ortholog, although nuclear staining was reported in early embryos.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000400 four-way junction DNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Binding of four-way (Holliday) junction DNA by GEN. Reason: DmGen binds Holliday junctions with sub-nanomolar affinity and dimerizes on them (EMSA, PMID:28369583); the IBA (GEN1 clade node) and ARBA rows agree. Junction binding is real but is the substrate-recognition step of the crossover junction endonuclease activity, which is the core function captured separately. Supporting Evidence: PMID:28369583 This analysis yields a K1/2,EMSA,HJ for binding of DmGen to the HJ0 equal to 0.19 ± 0.24 nM. |
| GO:0000400 four-way junction DNA binding | IDA PMID:28369583 Substrate preference of Gen endonucleases highlights the imp... | KEEP AS NON CORE | Summary: Binding of four-way (Holliday) junction DNA by GEN. Reason: DmGen binds Holliday junctions with sub-nanomolar affinity and dimerizes on them (EMSA, PMID:28369583); the IBA (GEN1 clade node) and ARBA rows agree. Junction binding is real but is the substrate-recognition step of the crossover junction endonuclease activity, which is the core function captured separately. Supporting Evidence: PMID:28369583 This analysis yields a K1/2,EMSA,HJ for binding of DmGen to the HJ0 equal to 0.19 ± 0.24 nM. |
| GO:0000400 four-way junction DNA binding | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Binding of four-way (Holliday) junction DNA by GEN. Reason: DmGen binds Holliday junctions with sub-nanomolar affinity and dimerizes on them (EMSA, PMID:28369583); the IBA (GEN1 clade node) and ARBA rows agree. Junction binding is real but is the substrate-recognition step of the crossover junction endonuclease activity, which is the core function captured separately. Supporting Evidence: PMID:28369583 This analysis yields a K1/2,EMSA,HJ for binding of DmGen to the HJ0 equal to 0.19 ± 0.24 nM. |
| GO:0000724 double-strand break repair via homologous recombination | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: double-strand break repair via homologous recombination (GEN1/Yen1 clade). Reason: Consistent with the PAINT node for the GEN1/Yen1 clade. In flies, Gen mutants are hypersensitive to ionizing radiation with MUS81 as backup, and synthetic lethality with Blm is partially suppressed by loss of Rad51 (spn-A), indicating that GEN processes recombination intermediates; its HJ and flap cleavage activities act directly on HR intermediates. Supporting Evidence: PMID:28369583 Gen mutants were significantly more sensitive to both agents, with mus81 mutants showing sensitivity only to HN2 PMID:22022278 Rather than dying at the first larval instar, the Gen mus309 spn-A mutants survive to the pupal stage |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro HhH2 domain mapping to DNA binding. Reason: Correct but general; GEN binds branched DNA, specifically four-way junctions (GO:0000400, already carried by the gene with IDA). Proposed replacements: four-way junction DNA binding Supporting Evidence: PMID:28369583 This analysis yields a K1/2,EMSA,HJ for binding of DmGen to the HJ0 equal to 0.19 ± 0.24 nM. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro mapping to catalytic activity. Reason: Correct but uninformative; GEN is a structure-selective endonuclease with experimentally shown 5'-flap and crossover junction (HJ resolvase) endonuclease activities, both already annotated. Proposed replacements: 5'-flap endonuclease activity crossover junction DNA endonuclease activity Supporting Evidence: PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 PMID:28369583 This junction allows us to determine if DmGen cuts symmetrically about the axis to produce nicked duplex products that can be ligated. PMID:28369583 Cleavage with DmGen creates nicked duplex DNA that can be ligated to create duplex DNA. |
| GO:0004518 nuclease activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro XPG-N/XPG-I mapping to nuclease activity. Reason: Correct but general; replace with the specific endonuclease activities demonstrated for DmGen. Proposed replacements: 5'-flap endonuclease activity crossover junction DNA endonuclease activity Supporting Evidence: PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 PMID:28369583 This junction allows us to determine if DmGen cuts symmetrically about the axis to produce nicked duplex products that can be ligated. PMID:28369583 Cleavage with DmGen creates nicked duplex DNA that can be ligated to create duplex DNA. |
| GO:0004520 DNA endonuclease activity | IDA PMID:15576351 DmGEN, a novel RAD2 family endo-exonuclease from Drosophila ... | MODIFY | Summary: DNA endonuclease activity from the first biochemical characterization of DmGEN. Reason: The original paper reported endonucleolytic cutting of DNA; follow-up work with an active-site mutant and with recombinant protein defined the activity as structure-selective cleavage of 5' flaps, replication-fork structures and Holliday junctions. The specific child terms are better. Proposed replacements: 5'-flap endonuclease activity crossover junction DNA endonuclease activity Supporting Evidence: PMID:15576351 In vitro, DmGEN showed endonuclease and 3'-5' PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 PMID:28369583 This junction allows us to determine if DmGen cuts symmetrically about the axis to produce nicked duplex products that can be ligated. PMID:28369583 Cleavage with DmGen creates nicked duplex DNA that can be ligated to create duplex DNA. |
| GO:0005634 nucleus | IDA PMID:17614965 DmGEN shows a flap endonuclease activity, cleaving the block... | KEEP AS NON CORE | Summary: Nuclear localization (immunostaining of early embryos; UniProt SubCell mapping of the same result). Reason: Kanai et al. 2007 detected DmGEN in nuclei of proliferating embryos, but a later study with a different antibody and tagged protein found DmGen largely or exclusively cytoplasmic in interphase, like human GEN1, which gains access to chromatin at nuclear envelope breakdown. GEN must act on nuclear DNA at some point, so the nuclear claim is plausible, but its timing and extent are contested; kept as non-core rather than overruling the curator. Supporting Evidence: PMID:17614965 revealed that DmGEN was present in the nucleus of actively proliferating PMID:28369583 While we cannot exclude the possibility that a low level of nuclear DmGen escaped our detection, our results imply that the genetic differences between DmGen and its orthologs are not simply due to differences in gross protein localization. file:DROME/Gen/Gen-deep-research-manual.md The two reports conflict; the more recent, better-controlled work favours interphase cytoplasmic sequestration |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Nuclear localization (immunostaining of early embryos; UniProt SubCell mapping of the same result). Reason: Kanai et al. 2007 detected DmGEN in nuclei of proliferating embryos, but a later study with a different antibody and tagged protein found DmGen largely or exclusively cytoplasmic in interphase, like human GEN1, which gains access to chromatin at nuclear envelope breakdown. GEN must act on nuclear DNA at some point, so the nuclear claim is plausible, but its timing and extent are contested; kept as non-core rather than overruling the curator. Supporting Evidence: PMID:17614965 revealed that DmGEN was present in the nucleus of actively proliferating PMID:28369583 While we cannot exclude the possibility that a low level of nuclear DmGen escaped our detection, our results imply that the genetic differences between DmGen and its orthologs are not simply due to differences in gross protein localization. file:DROME/Gen/Gen-deep-research-manual.md The two reports conflict; the more recent, better-controlled work favours interphase cytoplasmic sequestration |
| GO:0005737 cytoplasm | IDA PMID:28369583 Substrate preference of Gen endonucleases highlights the imp... | KEEP AS NON CORE | Summary: Cytoplasmic localization in embryos and S2 cells. Reason: Well-controlled immunofluorescence plus His-tagged expression show interphase cytoplasmic localization, consistent with regulatory sequestration seen for HsGEN1/Yen1. This is a location where GEN is held, not where its DNA-cleavage function is executed. Supporting Evidence: PMID:28369583 These results strongly suggest that our antibody binds to DmGen in cells and that DmGen, like Yen1 and HsGEN1, is primarily or exclusively cytoplasmic during interphase. |
| GO:0006281 DNA repair | IGI PMID:22022278 Three structure-selective endonucleases are essential in the... | ACCEPT | Summary: DNA repair from Gen mutant hypersensitivity to DNA-damaging agents and genetic interactions with Blm. Reason: Gen mutants are severely hypersensitive to MMS, HN2, camptothecin, hydroxyurea and IR, more so than mus81 mutants, and Gen is synthetically lethal with Blm (mus309). Combined with its structure-selective nuclease activity on repair intermediates, GEN directly participates in DNA repair. More specific processes (HR, replication fork processing) are captured on IBA rows. Supporting Evidence: PMID:28369583 Here, we confirm that DmGen is important in DNA damage repair by showing that, unlike in yeast and mammalian cells, Gen single mutants are severely hypersensitive to several different DNA damaging agents. PMID:22022278 Gen mutants have more severe hypersensitivities than mus81 mutants, suggesting that GEN plays a more critical role in DNA repair in Drosophila PMID:22022278 In support of this conclusion, Gen mus309 mutants are inviable, and die much earlier in development than either mus81; mus309 or mu312 mus309 double mutants. |
| GO:0006281 DNA repair | IMP PMID:22022278 Three structure-selective endonucleases are essential in the... | ACCEPT | Summary: DNA repair from Gen mutant hypersensitivity to DNA-damaging agents and genetic interactions with Blm. Reason: Gen mutants are severely hypersensitive to MMS, HN2, camptothecin, hydroxyurea and IR, more so than mus81 mutants, and Gen is synthetically lethal with Blm (mus309). Combined with its structure-selective nuclease activity on repair intermediates, GEN directly participates in DNA repair. More specific processes (HR, replication fork processing) are captured on IBA rows. Supporting Evidence: PMID:28369583 Here, we confirm that DmGen is important in DNA damage repair by showing that, unlike in yeast and mammalian cells, Gen single mutants are severely hypersensitive to several different DNA damaging agents. PMID:22022278 Gen mutants have more severe hypersensitivities than mus81 mutants, suggesting that GEN plays a more critical role in DNA repair in Drosophila PMID:22022278 In support of this conclusion, Gen mus309 mutants are inviable, and die much earlier in development than either mus81; mus309 or mu312 mus309 double mutants. |
| GO:0006281 DNA repair | IMP PMID:28369583 Substrate preference of Gen endonucleases highlights the imp... | ACCEPT | Summary: DNA repair from Gen mutant hypersensitivity to DNA-damaging agents and genetic interactions with Blm. Reason: Gen mutants are severely hypersensitive to MMS, HN2, camptothecin, hydroxyurea and IR, more so than mus81 mutants, and Gen is synthetically lethal with Blm (mus309). Combined with its structure-selective nuclease activity on repair intermediates, GEN directly participates in DNA repair. More specific processes (HR, replication fork processing) are captured on IBA rows. Supporting Evidence: PMID:28369583 Here, we confirm that DmGen is important in DNA damage repair by showing that, unlike in yeast and mammalian cells, Gen single mutants are severely hypersensitive to several different DNA damaging agents. PMID:22022278 Gen mutants have more severe hypersensitivities than mus81 mutants, suggesting that GEN plays a more critical role in DNA repair in Drosophila PMID:22022278 In support of this conclusion, Gen mus309 mutants are inviable, and die much earlier in development than either mus81; mus309 or mu312 mus309 double mutants. |
| GO:0006302 double-strand break repair | IMP PMID:17660539 A genetic screen for DNA double-strand break repair mutation... | ACCEPT | Summary: Double-strand break repair, from an I-SceI repair screen in which mus324 (= Gen) reduced SSA repair. Reason: The screen tested mus324, later shown to be a Gen allele (PMID:22022278); the effect on SSA was modest and condition-dependent, but a role of GEN in DSB repair is corroborated by IR hypersensitivity of Gen mutants (PMID:28369583) and the GEN1-clade HR annotation. Supporting Evidence: PMID:17660539 and mus324 behaved similarly in reducing SSA efficiency only with the milder 32° heat shock PMID:22022278 We conclude that mus324 is Gen PMID:28369583 Gen mutants were significantly more sensitive to both agents, with mus81 mutants showing sensitivity only to HN2 |
| GO:0008310 single-stranded DNA 3'-5' DNA exonuclease activity | IDA PMID:15576351 DmGEN, a novel RAD2 family endo-exonuclease from Drosophila ... | REMOVE | Summary: 3'-5' exonuclease activity reported in the first purification of DmGEN. Reason: UniProt curators judge this activity probably artifactual (contaminating nucleases in the preparation). The follow-up study from the same group, using the E143A/E145A active-site mutant as control, reports only weak nick-dependent 5'-3' exonuclease, and recombinant DmGen shows no cleavage of unbranched or nicked duplex DNA. 3'-5' exonuclease is also not an activity of the XPG/RAD2 family, whose members act on 5' structures. The claim is contradicted rather than unverifiable. Supporting Evidence: file:DROME/Gen/Gen-uniprot.txt 3' to 5' exonuclease activity reported in PubMed:15576351 is probably artifactual, due to the presence of other nucleases in the preparation. PMID:28369583 We did not detect cleavage of unbranched dsDNA, nicked duplex DNA |
| GO:0008311 double-stranded DNA 3'-5' DNA exonuclease activity | IDA PMID:15576351 DmGEN, a novel RAD2 family endo-exonuclease from Drosophila ... | REMOVE | Summary: 3'-5' exonuclease activity reported in the first purification of DmGEN. Reason: UniProt curators judge this activity probably artifactual (contaminating nucleases in the preparation). The follow-up study from the same group, using the E143A/E145A active-site mutant as control, reports only weak nick-dependent 5'-3' exonuclease, and recombinant DmGen shows no cleavage of unbranched or nicked duplex DNA. 3'-5' exonuclease is also not an activity of the XPG/RAD2 family, whose members act on 5' structures. The claim is contradicted rather than unverifiable. Supporting Evidence: file:DROME/Gen/Gen-uniprot.txt 3' to 5' exonuclease activity reported in PubMed:15576351 is probably artifactual, due to the presence of other nucleases in the preparation. PMID:28369583 We did not detect cleavage of unbranched dsDNA, nicked duplex DNA |
| GO:0008821 crossover junction DNA endonuclease activity | IDA PMID:28369583 Substrate preference of Gen endonucleases highlights the imp... | ACCEPT | Summary: Holliday junction (crossover junction) endonuclease activity. Reason: Recombinant DmGen cleaves fixed, mobile and nicked Holliday junctions symmetrically, producing ligatable nicked duplexes, the canonical HJ resolvase activity shared with HsGEN1/Yen1. This supersedes the 2007 report (reflected in the UniProt FUNCTION text) that DmGEN does not cleave HJs. Core MF. Supporting Evidence: PMID:28369583 This junction allows us to determine if DmGen cuts symmetrically about the axis to produce nicked duplex products that can be ligated. PMID:28369583 Cleavage with DmGen creates nicked duplex DNA that can be ligated to create duplex DNA. |
| GO:0008821 crossover junction DNA endonuclease activity | IEA GO_REF:0000117 | ACCEPT | Summary: Holliday junction (crossover junction) endonuclease activity. Reason: Recombinant DmGen cleaves fixed, mobile and nicked Holliday junctions symmetrically, producing ligatable nicked duplexes, the canonical HJ resolvase activity shared with HsGEN1/Yen1. This supersedes the 2007 report (reflected in the UniProt FUNCTION text) that DmGEN does not cleave HJs. Core MF. Supporting Evidence: PMID:28369583 This junction allows us to determine if DmGen cuts symmetrically about the axis to produce nicked duplex products that can be ligated. PMID:28369583 Cleavage with DmGen creates nicked duplex DNA that can be ligated to create duplex DNA. |
| GO:0017108 5'-flap endonuclease activity | IBA GO_REF:0000033 | ACCEPT | Summary: 5'-flap endonuclease activity. Reason: Shown with active-site mutant controls (PMID:17614965) and with recombinant protein, where 5' flaps are cleaved much faster than HJs (PMID:28369583); consistent with the XPG/RAD2 family IBA node. Core MF. Supporting Evidence: PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 |
| GO:0017108 5'-flap endonuclease activity | IDA PMID:28369583 Substrate preference of Gen endonucleases highlights the imp... | ACCEPT | Summary: 5'-flap endonuclease activity. Reason: Shown with active-site mutant controls (PMID:17614965) and with recombinant protein, where 5' flaps are cleaved much faster than HJs (PMID:28369583); consistent with the XPG/RAD2 family IBA node. Core MF. Supporting Evidence: PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 |
| GO:0017108 5'-flap endonuclease activity | IEA GO_REF:0000117 | ACCEPT | Summary: 5'-flap endonuclease activity. Reason: Shown with active-site mutant controls (PMID:17614965) and with recombinant protein, where 5' flaps are cleaved much faster than HJs (PMID:28369583); consistent with the XPG/RAD2 family IBA node. Core MF. Supporting Evidence: PMID:17614965 DmGEN had flap endonuclease activity PMID:28369583 Both full-length and truncated C-terminal His-tagged DmGen exhibit robust cleavage of 5 |
| GO:0031297 replication fork processing | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: replication fork processing (GEN1/Yen1 clade). Reason: DmGEN cleaves model replication forks (lagging strand) in vitro, and Gen mutants are strongly hypersensitive to replication-blocking agents (CPT, MMS, HU) with MUS81 acting as backup. GEN thus does work on stalled/regressed fork structures; consistent with the PAINT node. Supporting Evidence: PMID:17614965 cleaved the lagging strand of the model replication fork PMID:28369583 To investigate effects on replication-associated damage, we used (a) camptothecin (CPT), a topoisomerase I poison that results in replication-associated DSBs |
| GO:0035312 5'-3' DNA exonuclease activity | IDA PMID:15576351 DmGEN, a novel RAD2 family endo-exonuclease from Drosophila ... | KEEP AS NON CORE | Summary: Weak nick-dependent 5'-3' exonuclease activity. Reason: Reported in the original study and confirmed as weak, nick-dependent activity with an active-site mutant control (PMID:17614965); Bellendir et al. 2017 did not detect cleavage of nicked duplex DNA. A minor, FEN1-like side activity, not the core function. Supporting Evidence: PMID:15576351 5'-3' exonuclease activity was found to occur with nicked DNA |
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Download this section (compressed HTML)Q: Does GEN resolve Holliday junctions in vivo in Drosophila (e.g. contribute to mitotic crossovers or to resolution of recombination intermediates, GO:0071140), or are 5' flaps and simpler branched structures its main physiological substrates, as its much faster flap cleavage suggests?
Q: How is GEN access to chromatin regulated in flies: is it excluded from interphase nuclei and released at nuclear envelope breakdown as for human GEN1, and how can the earlier nuclear immunostaining in embryos be reconciled?
Q: The UniProt Q9VRJ0 FUNCTION text states that DmGEN does not cleave Holliday junction substrates; should it be updated in light of PMID:28369583?
Experiment: Image endogenously tagged GEN through syncytial and post-blastoderm cell cycles and in larval neuroblasts, with and without DNA damage.
Hypothesis: GEN is cytoplasmic in interphase and gains access to chromosomes at mitosis.
Type: Live imaging / cell-cycle localization
Experiment: Rescue Gen Blm double mutants with transgenes carrying catalytic-dead or dimerization-defective GEN that retains flap but not HJ cleavage, and score viability and chromosome breaks.
Hypothesis: GEN HJ-cleavage, rather than flap cleavage, underlies viability of Blm mutants.
Type: Separation-of-function genetics
XPG-like endonuclease (Gen) encodes a nuclease that cuts Holliday junctions, flaps, and replication forks during DNA and replication fork repair.
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