Rad9 encodes the Drosophila ortholog of RAD9, the subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp that carries the C-terminal tail used to direct the complex and engage checkpoint activators. DmRad9 interacts directly with Hus1 and co-precipitates with Rad1 and Hus1. Two splice forms differ at the C terminus. DmRad9B is nuclear, whereas DmRad9A uses a 32-residue C-terminal extension to target the 9-1-1 complex to the nuclear envelope. DmRad9A localization is altered when the meiotic checkpoint is activated by DNA repair defects, suggesting a role at the nuclear periphery in the meiotic DNA damage response. Rad9 associates with Brca2, and Rad9 mutant females show persistent meiotic DSB signals and karyosome defects, with the Rad9B isoform being most effective at restoring efficient early meiotic break repair.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000077 DNA damage checkpoint signaling | IEA GO_REF:0000002 | ACCEPT | Summary: The 9-1-1 clamp is a component of the DNA integrity checkpoint system. Reason: Conserved function of the 9-1-1 clamp, which is loaded at damaged DNA and helps activate ATR (MEI-41) signaling; supported in Drosophila by the hus1 mutant phenotypes (HU/MMS sensitivity, loss of the meiotic checkpoint). Supporting Evidence: PMID:22666434 Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. PMID:17327271 These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint |
| GO:0000077 DNA damage checkpoint signaling | NAS PMID:22666434 Localization of the Drosophila Rad9 protein to the nuclear m... | ACCEPT | Summary: The 9-1-1 clamp is a component of the DNA integrity checkpoint system. Reason: Conserved function of the 9-1-1 clamp, which is loaded at damaged DNA and helps activate ATR (MEI-41) signaling; supported in Drosophila by the hus1 mutant phenotypes (HU/MMS sensitivity, loss of the meiotic checkpoint). Supporting Evidence: PMID:22666434 Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. PMID:17327271 These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint |
| GO:0005634 nucleus | IDA PMID:22666434 Localization of the Drosophila Rad9 protein to the nuclear m... | ACCEPT | Summary: DmRad9 (isoform B and the shared NLS) localizes to the nucleus. Reason: Direct localization data in Drosophila cells; Rad9 carries the NLS of the 9-1-1 complex. Supporting Evidence: PMID:22666434 DmRad9B is localized at the cell nucleus |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: DmRad9 (isoform B and the shared NLS) localizes to the nucleus. Reason: Direct localization data in Drosophila cells; Rad9 carries the NLS of the 9-1-1 complex. Supporting Evidence: PMID:22666434 DmRad9B is localized at the cell nucleus |
| GO:0005635 nuclear envelope | EXP PMID:22666434 Localization of the Drosophila Rad9 protein to the nuclear m... | ACCEPT | Summary: The 9-1-1 complex containing Rad9 isoform A localizes to the nuclear membrane in S2 cells and follicle cells. Reason: Localization of tagged proteins in Drosophila cells and tissues; Rad9A's C-terminal region targets the complex to the nuclear periphery, and this localization changes upon activation of the meiotic checkpoint. Supporting Evidence: PMID:22666434 DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells |
| GO:0005635 nuclear envelope | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping of isoform A to the nuclear envelope. Reason: DmRad9A localizes to the nuclear membrane via its specific C-terminal residues. Supporting Evidence: PMID:22666434 the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane |
| GO:0006281 DNA repair | IEA GO_REF:0000002 | ACCEPT | Summary: DNA repair mapped from the RAD9 family InterPro signature. Reason: The 9-1-1 clamp contributes to DNA repair; DmRad9 interacts with Brca2 and the Hus1 subunit is required for meiotic HR repair. Supporting Evidence: file:DROME/Rad9/Rad9-uniprot.txt Interacts with Brca2 file:DROME/Rad9/Rad9-deep-research-falcon.md Thus Rad9B provided the convincing rescue of efficient early meiotic break resolution in these assays PMID:19501158 Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination. |
| GO:0008296 3'-5'-DNA exonuclease activity | ISS GO_REF:0000024 | MARK AS OVER ANNOTATED | Summary: 3'-5' exonuclease activity transferred by sequence similarity from early reports on mammalian RAD9. Reason: Rad9 is a PCNA-like clamp subunit; there is no Drosophila evidence of nuclease activity and no catalytic nuclease domain is annotated in the fly protein (UniProt places it in the rad9 family only). The ISS transfer extends a contested in vitro activity of the mammalian protein to a clamp subunit whose established role is structural. Supporting Evidence: file:DROME/Rad9/Rad9-uniprot.txt FUNCTION: Component of the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp complex. |
| GO:0030896 checkpoint clamp complex | IDA PMID:22666434 Localization of the Drosophila Rad9 protein to the nuclear m... | ACCEPT | Summary: Co-immunoprecipitation of tagged DmRad9, DmRad1 and DmHus1 from S2 cells shows that the three proteins form the Drosophila 9-1-1 checkpoint clamp. Reason: Direct biochemical evidence in Drosophila cells, consistent with the conserved Rad9-Rad1-Hus1 heterotrimeric clamp; the IBA and InterPro-based annotations agree. Supporting Evidence: PMID:22666434 Our results demonstrate for the first time that it is possible to co-precipitate DmRad9 with DmRad1 (Figure 3) and Hus1, indicating that DmRad9 forms a complex with DmRad1 and DmHus1. |
| GO:0030896 checkpoint clamp complex | IEA GO_REF:0000002 | ACCEPT | Summary: Co-immunoprecipitation of tagged DmRad9, DmRad1 and DmHus1 from S2 cells shows that the three proteins form the Drosophila 9-1-1 checkpoint clamp. Reason: Direct biochemical evidence in Drosophila cells, consistent with the conserved Rad9-Rad1-Hus1 heterotrimeric clamp; the IBA and InterPro-based annotations agree. Supporting Evidence: PMID:22666434 Our results demonstrate for the first time that it is possible to co-precipitate DmRad9 with DmRad1 (Figure 3) and Hus1, indicating that DmRad9 forms a complex with DmRad1 and DmHus1. |
| GO:0030896 checkpoint clamp complex | IPI PMID:22666434 Localization of the Drosophila Rad9 protein to the nuclear m... | ACCEPT | Summary: Co-immunoprecipitation of tagged DmRad9, DmRad1 and DmHus1 from S2 cells shows that the three proteins form the Drosophila 9-1-1 checkpoint clamp. Reason: Direct biochemical evidence in Drosophila cells, consistent with the conserved Rad9-Rad1-Hus1 heterotrimeric clamp; the IBA and InterPro-based annotations agree. Supporting Evidence: PMID:22666434 Our results demonstrate for the first time that it is possible to co-precipitate DmRad9 with DmRad1 (Figure 3) and Hus1, indicating that DmRad9 forms a complex with DmRad1 and DmHus1. |
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Download this section (compressed HTML)Q: Does the Drosophila Rad9 C-terminal tail contain a TopBP1 (mus101)-binding motif needed to activate MEI-41, and do the two splice isoforms differ in checkpoint activity?
Q: Is the reported 3'-5' exonuclease activity of RAD9 intrinsic to the protein, or should the ISS transfer to Drosophila be withdrawn?
Experiment: Generate isoform-specific Rad9 alleles and test meiotic checkpoint activation (Vasa modification, dorsal-ventral patterning) in DSB-repair mutant backgrounds.
Hypothesis: The nuclear-envelope-targeted Rad9A isoform is specifically required for the meiotic checkpoint.
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Download this section (compressed HTML)Rad9 (Rad9) encodes a protein that, together with the Rad1 and Hus1-like products, forms the 9-1-1 checkpoint protein complex. This complex plays a central role in the DNA damage-induced checkpoint response.
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