id: MODULE:atm_dsb_signaling title: ATM signaling at DNA double-strand breaks — MRN sensing and ATM activation, gammaH2AX-MDC1 amplification, the RNF8-UBE2N-RNF168 ubiquitin cascade, 53BP1-RIF1 versus BRCA1-A recruitment, and phosphatase/deubiquitinase/HECT-ligase attenuation; ATM/MRE11/RAD50/NBN/KAT5/H2AX/MDC1/RNF8/UBE2N/RNF168/TP53BP1/RIF1/BRCA1 description: >- A DNA double-strand break (DSB) in chromatin is detected by the MRE11-RAD50-NBN (MRN) complex, which binds and tethers the broken DNA ends and recruits the serine/threonine kinase ATM through a conserved C-terminal motif of NBN. Inactive ATM dimers dissociate into active monomers that autophosphorylate on Ser1981, and acetylation of ATM by the histone acetyltransferase KAT5 (TIP60), which is held in a constitutive complex with ATM, is needed for full kinase activity. Active ATM phosphorylates hundreds of substrates. At the break it phosphorylates the histone variant H2AX on Ser139 over megabase domains of flanking chromatin, producing gammaH2AX. The tandem BRCT domain of MDC1 binds the phosphorylated H2AX C-terminus, and MDC1 in turn binds ATM and NBN, concentrating more MRN-ATM on the flanking chromatin and so spreading H2AX phosphorylation in a positive feedback loop. ATM-phosphorylated MDC1 recruits the RING E3 ubiquitin ligase RNF8 through its FHA domain. RNF8, working with the E2 enzyme UBE2N (UBC13), initiates K63-linked ubiquitin chains on chromatin proteins, which recruit a second RING ligase, RNF168. RNF168 monoubiquitylates nucleosomal H2A and H2AX on Lys13/Lys15 and extends K63-linked chains, amplifying the ubiquitin signal. These marks recruit two antagonistic readers. 53BP1 (TP53BP1) binds nucleosomes that carry both H4K20me2 and H2AK15ub; the H4K20me2 sites are first unmasked by VCP/p97-driven extraction of L3MBTL1 and by RNF8/RNF168-dependent degradation of KDM4A. ATM-phosphorylated 53BP1 recruits RIF1, and together they block DNA end resection and favour non-homologous end joining, chiefly in G1. The BRCA1-A complex, recruited by the RAP80 (UIMC1) ubiquitin-interaction motifs to K63 chains, carries BRCA1-BARD1 and the K63-specific deubiquitinase BRCC3 (BRCC36); BRCA1 and its partner CtIP displace 53BP1-RIF1 from S phase onward, and KAT5 acetylation of H2AK15, which blocks its ubiquitylation, further limits 53BP1 binding, so that resection and homologous recombination are favoured. SUMO conjugation by PIAS1 and PIAS4 is needed for productive accumulation of 53BP1, BRCA1 and RNF168, and the SUMO-targeted ubiquitin ligase RNF4 turns over SUMOylated MDC1. In parallel, ATM-phosphorylated RNF20-RNF40 monoubiquitylates H2B to relax chromatin for repair. The phosphorylation state of H2AX Tyr142, set by the WSTF (BAZ1B) tyrosine kinase and removed by EYA phosphatases, gates MDC1 binding and the choice between repair and apoptosis. The signal is confined and switched off by phosphatases that dephosphorylate ATM and gammaH2AX (PP2A, PP4, PP6 and WIP1/PPM1D), by deubiquitinases that remove the chromatin ubiquitin marks (USP3, USP16, USP44) or inhibit UBE2N without catalysis (OTUB1), and by the HECT ligases TRIP12 and UBR5, which keep RNF168 levels limiting. ATM output then drives checkpoint arrest through CHK2 and p53, apoptosis or senescence, and the repair reactions themselves; those downstream programs are outside this module. status: DRAFT scope: CONCRETE evidence: - source_id: PW:0001361 title: ataxia telangiectasia-mutated (ATM) signaling pathway statement: >- The module boundary follows the RGD Pathway Ontology term and its diagram. The diagram draws Atm associated with the MRN complex and with Mdc1; Kat5, Mdc1 and the "ATM/DSB regulators" box positively affecting Atm; Atm positively affecting H2afx, MRN, Mdc1, Brca1, Tp53bp1, Rnf20 and Rnf40; Baz1b positively affecting H2afx (PY142); H2afx associated with Mdc1; Mdc1 associated with Rnf8; Rif1 and Mum1 associated with Tp53bp1; Brca1 associated with Rbbp8 and antagonising Tp53bp1; and Tp53bp1 and Brca1 driving the NHEJ and HR subpathways. Derived through projects/RGD_PATHWAY_ONTOLOGY (pathways/PW_0001361.yaml). - source_id: GO:0042770 title: signal transduction in response to DNA damage statement: >- Curated skos:broadMatch for PW:0001361 in projects/RGD_PATHWAY_ONTOLOGY/data/pw_go_curated.sssom.yaml. GO has no DSB-specific or ATM-specific signalling term, so this is the module's process concept. - source_id: GO:0000077 title: DNA damage checkpoint signaling statement: >- Curated skos:narrowMatch for PW:0001361. It covers only the checkpoint arm of ATM signalling, and it also covers ATR-dependent checkpoint signalling, so it is recorded as a related process on the ATM annoton rather than as the module concept. - source_id: gomodel:617b481400000460 title: KAT5 involvement in DNA repair via homologous recombination (Human) statement: >- The project's best-overlap GO-CAM for PW:0001361 (cached under gocams/617b481400000460/). It models RNF168 histone H2AK15 ubiquitin ligase activity directly providing input for TP53BP1 ubiquitin-modified histone reader and H4K20me2 reader activities, and KAT5 histone H2A acetyltransferase and the MBTD1 H4K20me reader acting on the 53BP1 node, plus PIAS4 SUMO ligase activity. The pathway-choice part of this module is grounded in it. - source_id: gomodel:65a1f4f800000487 title: Phosphorylation of H2AX by ATM or ATR promotes recruitment of NBN and MRN complex to DNA damage sites (Human). statement: >- Human GO-CAM in which ATM (and ATR) histone H2AXS139 kinase activity directly provides input for H2AX chromatin-protein adaptor activity, which positively regulates NBN chromatin-protein adaptor activity. It is the basis for treating H2AX as a participant (scaffold) rather than only a substrate. - source_id: PMID:15790808 title: ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. statement: MRN is the DSB sensor that recruits and activates ATM. supporting_text: We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. - source_id: PMID:16427009 title: MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. statement: H2AX, MDC1 and ATM form the positive feedback loop at the core of the module. supporting_text: At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. - source_id: PMID:23333306 title: A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. statement: The module ends at the 53BP1 versus BRCA1 pathway-choice node. supporting_text: DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. notes: >- Derived from the RGD Pathway Ontology diagram PW:0001361 through the RGD_PATHWAY_ONTOLOGY project's diagram->module route (seed in projects/RGD_PATHWAY_ONTOLOGY/data/module_seeds.yaml, tier candidate; no existing module). RGD curates 39 human genes to PW:0001361: ATM BAZ1B BRCA1 BRCC3 EYA1 EYA3 H2AX KAT5 KDM4A L3MBTL1 MDC1 MRE11 NBN OTUB1 PIAS1 PIAS4 PPM1D PPP2CA PPP4C PPP5C PPP6C PSMD14 PWWP3A RAD50 RBBP8 RIF1 RNF168 RNF20 RNF4 RNF40 RNF8 TP53BP1 TRIP12 UBE2N UBR5 USP16 USP3 USP44 VCP. Thirty-four are modelled as participants (KDM4A and L3MBTL1 appear only as targets of the activities that remove them; see decision 4). Five gene products not on the RGD list are added as units of the BRCA1-A complex (UIMC1/RAP80, ABRAXAS1, BABAM1, BABAM2, BARD1), because the RGD list includes BRCC3 and BRCA1 but not the subunits that make the complex a ubiquitin reader. Molecular functions were taken from each protein's experimental GO annotations in QuickGO (October 2026) and, where a gene review exists (ATM, BRCA1, VCP, EYA1, PIAS1, PPP2CA, ABRAXAS1, BABAM1, BABAM2, BARD1), from its core functions. Two departures from experimental human GOA are deliberate: ATM histone H2AXS139 kinase activity (GO:0035979) has only ISS/IEA rows in human (accepted in the ATM review), but it is the activity the curated human GO-CAM gomodel:65a1f4f800000487 uses and is supported by PMID:11571274; and RIF1 has no experimental MF row, so its annoton carries a process but no function. Comparator check for H2AX (CLAUDE.md, substrate versus participant). H2AX is phosphorylated by ATM, ubiquitylated by RNF8/RNF168 and dephosphorylated by PPM1D/PP2A/PP4/PP6, so it is a substrate several times over. It is nevertheless modelled as a participant, because it does work that the downstream steps depend on: the phosphorylated C-terminal tail is the docking epitope for MDC1, so H2AX is the scaffold case (like thyroglobulin in thyroid hormone generation) rather than the angiotensinogen case. Curators agree: H2AX carries chromatin-protein adaptor activity GO:0140463 (IDA), GO:1990166 protein localization to site of double-strand break (IDA) and GO:0000077 DNA damage checkpoint signaling (IDA), and the human GO-CAM gomodel:65a1f4f800000487 gives H2AX its own adaptor activity that positively regulates NBN. The other human proteins annotated to GO:1990166 in QuickGO include MDC1, NBN and TP53BP1, the scaffold/reader members of this module. By contrast, KDM4A and L3MBTL1 are only objects of the cascade: they are H4K20me2 readers that occlude 53BP1 binding sites and are removed by RNF8/RNF168-dependent degradation (KDM4A) or VCP-driven extraction (L3MBTL1). Their role is inhibitory and passive, so they appear as targets of the RNF168 and VCP activities rather than as annotons. Boundary decisions. (1) The module stops at the recruitment of the two pathway-choice readers. Checkpoint effectors (CHK2, p53, CDC25A), apoptosis and senescence, end resection (RBBP8/CtIP, EXO1, DNA2-BLM), homologous recombination (BRCA2, PALB2, RAD51) and non-homologous end joining (Ku, DNA-PKcs, XRCC4-LIG4, shieldin) are downstream programs. When this module was written no human module existed for any of them (modules/ was searched for p53, checkpoint, homologous recombination, NHEJ, resection, MRN and ATM). They are now modelled in modules/p53_signaling.yaml, modules/homologous_recombination_dsb_repair.yaml and modules/nonhomologous_end_joining.yaml, which refer back to this module's parts. Otherwise the only related human-scope node is g2_m_transition#checkpoint_restraint, an abstract CHK1-centred step that is the ATR rather than the ATM arm, so it is not referenced. Other-taxon modules on neighbouring biology exist only by name: dmel_mrn_complex, dmel_atr_atrip_complex, dmel_9_1_1_checkpoint_clamp, bacterial_nonhomologous_end_joining and bacterial_recbcd_end_resection. The PW subpathways RGD links from the diagram (PW:0000094 cell cycle checkpoint, PW:0000718 p53 signaling, PW:0000009 apoptotic cell death, PW:0000202 homologous recombination, PW:0000203 non-homologous end joining, PW:0000235 adaptive immune response via V(D)J and class switch recombination) are therefore mentioned here but not listed as evidence sources. (2) RBBP8 (CtIP) is excluded as a participant. It is on the RGD list and associated with Brca1 on the diagram, but its activity (single-stranded DNA endonuclease, with MRE11 in resection) is the first step of the HR reaction, not of the signal; it is named in the BRCA1 variant as the partner through which BRCA1 antagonises 53BP1-RIF1 (PMID:23333306). (3) PWWP3A (MUM1/EXPAND1) is excluded. RGD draws Mum1 associated with Tp53bp1 and its only experimental MF in human GOA is nucleosome binding; its role as a 53BP1-dependent chromatin decondensation factor rests on a single study not cached here, and it is recorded as a candidate to add to the 53BP1 arm. (4) KDM4A and L3MBTL1 are targets, not participants (see the comparator paragraph). (5) PSMD14 (POH1/RPN11) is excluded. It is the constitutive deubiquitinase of the 19S proteasome lid; it opposes RNF8/RNF168 K63 chains and restricts 53BP1 accumulation (PMID:22909820), but this is proteasome function acting on the DSB ubiquitin marks, and it is better placed in a proteasome module than as a dedicated signalling component. (6) PPP5C (PP5) is placed with ATM activation, not with attenuation: although RGD groups it with the phosphatases, PP5 is required for DNA-damage-induced ATM autophosphorylation and activity (PMID:14871926). (7) BAZ1B (WSTF), EYA1 and EYA3 are included as the H2AX Tyr142 switch, because Y142 dephosphorylation is required for MDC1 binding and so gates the main amplification loop; the apoptosis branch that pY142 favours is left downstream. BAZ1B's chromatin-remodelling role in the WICH complex is not modelled. (8) RNF20-RNF40 H2B monoubiquitylation is included as a parallel, H2AX-independent ATM output, as RGD draws it (Atm --+> Rnf20/Rnf40 --+> chromatin relaxation). (9) The SUMO arm (PIAS1, PIAS4, RNF4) is included because PIAS-dependent SUMOylation is required for productive 53BP1, BRCA1 and RNF168 accumulation and RNF4 clears SUMO-MDC1; it is in the KAT5 GO-CAM (gomodel:617b481400000460, PIAS4) and the MRE11 SUMOylation GO-CAM (gomodel:65a1f4f800002665, PIAS1). RNF4 and VCP also act together in the PARP1 extraction GO-CAM gomodel:627d95ee00001139, a different process not used here. (10) Effector checkpoint kinase CHK2 and p53 are not modelled; ATM's process annotation to GO:0000077 records the checkpoint output. GO-CAM and GOA observations for upstream review. In gomodel:617b481400000460 the KAT5 activity at node 617b481400000763 is typed as bare protein binding (GO:0005515); the model's own evidence (PMID:27153538) supports KAT5 H2AK15 acetylation, already captured at node 617b481400000461. In gomodel:65a1f4f800000487 H2AX positively regulates NBN directly, without MDC1, although MDC1 is the reader of gammaH2AX that recruits NBN (PMID:16427009); the companion model gomodel:65a1f4f800000428 includes MDC1. ATM carries GO:0035979 only by ISS/IEA in human GOA despite direct in vitro evidence (PMID:11571274). RIF1 has no experimental molecular function in human GOA. Gene reviews exist for ATM, BRCA1, BARD1, VCP, EYA1, PIAS1, PPP2CA, ABRAXAS1, BABAM1 and BABAM2; the other members have none. PANTHER family ids and PTNs are deliberately not asserted. module: id: atm_dsb_signaling label: ATM signaling at DNA double-strand breaks module_type: SIGNALING_PATHWAY concepts: - preferred_term: signal transduction in response to DNA damage term: id: GO:0042770 label: signal transduction in response to DNA damage description: >- The GO process mapped (skos:broadMatch) to PW:0001361. Here restricted to the ATM branch initiated by double-strand breaks. - preferred_term: protein localization to site of double-strand break term: id: GO:1990166 label: protein localization to site of double-strand break description: >- The assembly of mediator and reader proteins on DSB-flanking chromatin that is the cascade's direct output. context: taxa: - preferred_term: Homo sapiens term: id: NCBITaxon:9606 label: Homo sapiens cellular_components: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break parts: - order: 1 role: DSB sensing by MRN and ATM activation node: id: atm_activation label: MRN recruitment and activation of ATM, with KAT5 acetylation of ATM module_type: REGULATORY_STEP description: >- MRN binds the DNA ends and recruits ATM through the NBN C-terminus; inactive ATM dimers dissociate, ATM autophosphorylates on Ser1981 and is acetylated by KAT5 (TIP60), and PP5 activity is required for full activation. annotons: - id: mrn_atm_activator label: MRN complex activates ATM at DNA ends participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: MRE11-RAD50-NBN (MRN) complex term: id: GO:0030870 label: Mre11 complex active_units: - id: mrn_mre11 label: MRE11 nuclease subunit role: Binds and processes DNA ends; nuclease core of the complex. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MRE11 term: id: UniProtKB:P49959 label: Double-strand break repair protein MRE11 function: preferred_term: DNA endonuclease activity term: id: GO:0004520 label: DNA endonuclease activity - id: mrn_rad50 label: RAD50 ATPase subunit role: ATP-dependent DNA end binding and tethering; DNA unwinding by MRN is needed for ATM stimulation. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RAD50 term: id: UniProtKB:Q92878 label: DNA repair protein RAD50 function: preferred_term: ATP hydrolysis activity term: id: GO:0016887 label: ATP hydrolysis activity - id: mrn_nbn label: NBN (NBS1) ATM-recruiting subunit role: Its conserved C-terminal motif binds and recruits ATM to the break. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: NBN term: id: UniProtKB:O60934 label: Nibrin function: preferred_term: protein serine/threonine kinase activator activity term: id: GO:0043539 label: protein serine/threonine kinase activator activity evidence: - source_id: PMID:15758953 statement: The NBN C-terminal motif is required for ATM recruitment to damage and for ATM signalling. supporting_text: Here we identify related, conserved carboxy-terminal motifs in human Nbs1, ATRIP and Ku80 proteins that are required for their interaction with ATM, ATR and DNA-PKcs, respectively. function: preferred_term: protein serine/threonine kinase activator activity term: id: GO:0043539 label: protein serine/threonine kinase activator activity targets: - preferred_term: ATM term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- The DSB sensor for ATM. MRN is also an ATM substrate, and hypomorphic NBN and MRE11 mutations cause Nijmegen breakage syndrome and A-T-like disease. evidence: - source_id: PMID:15790808 statement: MRN monomerises and activates ATM in vitro; DNA unwinding by MRN is essential. supporting_text: Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. - source_id: PMID:14532133 statement: Functional MRN is required for ATM activation in cells. supporting_text: Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. gocam_associations: - model: gomodel:65a1f4f800002560 activity: gomodel:65a1f4f800002560/65a1f4f800002570 title: Mre11 complex protein serine/threonine kinase activator activity (GO:0043539) - model: gomodel:65a1f4f800002665 activity: gomodel:65a1f4f800002665/65a1f4f800002675 title: Mre11 complex protein serine/threonine kinase activator activity (GO:0043539) - id: kat5_atm_acetylation label: KAT5 (TIP60) acetylation of ATM participant: selector_type: GENE_PRODUCT gene_product: preferred_term: KAT5 (TIP60) term: id: UniProtKB:Q92993 label: Histone acetyltransferase KAT5 function: preferred_term: protein-lysine-acetyltransferase activity term: id: GO:0061733 label: protein-lysine-acetyltransferase activity targets: - preferred_term: ATM term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Forms a stable complex with ATM through the ATM FATC domain; its acetyltransferase activity is switched on by DNA damage and is needed for ATM kinase activation. evidence: - source_id: PMID:16141325 statement: DNA damage induces Tip60-dependent ATM acetylation, and Tip60 loss blocks ATM activation. supporting_text: This acetylation depends on the Tip60 histone acetyltransferase (HAT). Suppression of Tip60 blocks the activation of ATM's kinase activity and prevents the ATM-dependent phosphorylation of p53 and chk2. - id: ppp5c_atm_activation label: PP5 (PPP5C) requirement for ATM activation participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PPP5C term: id: UniProtKB:P53041 label: Serine/threonine-protein phosphatase 5 function: preferred_term: protein serine/threonine phosphatase activity term: id: GO:0004722 label: protein serine/threonine phosphatase activity locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Binds ATM in a damage-inducible manner; catalytically inactive PP5 blocks ATM Ser1981 autophosphorylation. Its phosphatase substrate in this role is not known. evidence: - source_id: PMID:14871926 statement: Catalytic activity of PP5 is needed for ATM autophosphorylation and checkpoint signalling. supporting_text: Expression of a catalytically inactive PP5 mutant inhibited the phosphorylation of ATM substrates and the autophosphorylation of ATM on Ser 1981, and caused an S-phase checkpoint defect in DNA-damaged cells. - id: atm_kinase label: ATM serine/threonine kinase activity on DSB-response substrates participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ATM term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM function: preferred_term: protein serine/threonine kinase activity term: id: GO:0004674 label: protein serine/threonine kinase activity targets: - preferred_term: ATM (Ser1981 autophosphorylation) term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM - preferred_term: NBN term: id: UniProtKB:O60934 label: Nibrin - preferred_term: MDC1 term: id: UniProtKB:Q14676 label: Mediator of DNA damage checkpoint protein 1 - preferred_term: TP53BP1 term: id: UniProtKB:Q12888 label: TP53-binding protein 1 - preferred_term: BRCA1 term: id: UniProtKB:P38398 label: Breast cancer type 1 susceptibility protein - preferred_term: RNF20 term: id: UniProtKB:Q5VTR2 label: E3 ubiquitin-protein ligase BRE1A processes: - preferred_term: DNA damage checkpoint signaling term: id: GO:0000077 label: DNA damage checkpoint signaling locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- The pathway kinase. Activated ATM phosphorylates mediators at the break and soluble effectors (CHK2, p53) that carry the checkpoint output, which is outside the module. evidence: - source_id: PMID:12556884 statement: Intermolecular Ser1981 autophosphorylation dissociates inactive ATM dimers. supporting_text: Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. gocam_associations: - model: gomodel:65a1f4f800002560 activity: gomodel:65a1f4f800002560/65a1f4f800002582 title: ATM protein serine/threonine kinase activity (GO:0004674), Ufmylation of MRE11 promotes ATM activation by MRN (Human) - order: 2 role: H2AX Ser139 phosphorylation and gammaH2AX scaffold node: id: h2ax_phosphorylation label: ATM phosphorylation of H2AX Ser139 (gammaH2AX) module_type: REACTION description: >- ATM phosphorylates H2AX on Ser139 in chromatin flanking the break; the phosphorylated tail is the docking platform that organises the rest of the cascade. DNA-PK contributes in the absence of ATM. annotons: - id: atm_h2ax_kinase label: ATM histone H2AX Ser139 kinase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ATM term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM function: preferred_term: histone H2AXS139 kinase activity term: id: GO:0035979 label: histone H2AXS139 kinase activity targets: - preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: The major H2AX Ser139 kinase after DSBs. evidence: - source_id: PMID:11571274 statement: ATM phosphorylates H2AX in vitro and is required for H2AX phosphorylation and foci in cells. supporting_text: Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. gocam_associations: - model: gomodel:65a1f4f800000487 activity: gomodel:65a1f4f800000487/65a1f4f800000547 title: ATM histone H2AXS139 kinase activity (GO:0035979), directly provides input for H2AX - model: gomodel:65a1f4f800000428 activity: gomodel:65a1f4f800000428/65a1f4f800000480 title: ATM histone H2AXS139 kinase activity (GO:0035979) - id: h2ax_scaffold label: gammaH2AX chromatin-protein adaptor (MDC1 docking site) participant: selector_type: GENE_PRODUCT gene_product: preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX function: preferred_term: chromatin-protein adaptor activity term: id: GO:0140463 label: chromatin-protein adaptor activity processes: - preferred_term: protein localization to site of double-strand break term: id: GO:1990166 label: protein localization to site of double-strand break locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Substrate and scaffold: the phospho-Ser139 C-terminus is the epitope bound by the MDC1 BRCT repeat, which anchors the downstream mediators. Modelled as a participant for that reason (see notes). evidence: - source_id: PMID:16377563 statement: gammaH2AX phosphorylation is the main recruitment signal, read directly by MDC1. supporting_text: Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. gocam_associations: - model: gomodel:65a1f4f800000487 activity: gomodel:65a1f4f800000487/65a1f4f800000527 title: H2AX chromatin-protein adaptor activity (GO:0140463), part of GO:1990166 - order: 3 role: MDC1 reading of gammaH2AX and feed-forward amplification node: id: mdc1_amplification label: MDC1 binding to gammaH2AX and accumulation of MRN-ATM module_type: REGULATORY_STEP description: >- MDC1 binds gammaH2AX through its BRCT repeat and ATM through its FHA domain, retaining active ATM and MRN on the flanking chromatin and spreading H2AX phosphorylation; ATM-phosphorylated MDC1 TQXF motifs then recruit RNF8. annotons: - id: mdc1_gh2ax_reader label: MDC1 histone reader activity (gammaH2AX) participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MDC1 term: id: UniProtKB:Q14676 label: Mediator of DNA damage checkpoint protein 1 function: preferred_term: histone reader activity term: id: GO:0140566 label: histone reader activity targets: - preferred_term: gammaH2AX (H2AX phospho-Ser139) term: id: UniProtKB:P16104 label: Histone H2AX processes: - preferred_term: protein localization to site of double-strand break term: id: GO:1990166 label: protein localization to site of double-strand break locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Mediator and signal amplifier; Mdc1-null mice recapitulate many H2AX-null phenotypes. evidence: - source_id: PMID:16377563 statement: The MDC1 BRCT repeat binds the gammaH2AX phosphoepitope directly. supporting_text: These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. - source_id: PMID:16427009 statement: MDC1 binds gammaH2AX via its BRCT domain and ATM via its FHA domain, accumulating active ATM. supporting_text: MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. gocam_associations: - model: gomodel:65a1f4f800000428 activity: gomodel:65a1f4f800000428/65a1f4f800000472 title: MDC1 histone reader activity (GO:0140566), part of GO:1990166 - order: 4 role: H2AX Tyr142 phosphorylation switch gating MDC1 binding node: id: h2ax_y142_switch label: BAZ1B (WSTF) phosphorylation and EYA1/EYA3 dephosphorylation of H2AX Tyr142 module_type: REGULATORY_STEP description: >- H2AX Tyr142 is constitutively phosphorylated by the WSTF (BAZ1B) tyrosine kinase. After damage, EYA phosphatases remove pY142, which favours MDC1 binding to gammaH2AX and repair; doubly phosphorylated (pS139/pY142) H2AX instead recruits pro-apoptotic factors. annotons: - id: baz1b_y142_kinase label: BAZ1B (WSTF) histone H2AX Tyr142 kinase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BAZ1B (WSTF) term: id: UniProtKB:Q9UIG0 label: Tyrosine-protein kinase BAZ1B function: preferred_term: histone H2AXY142 kinase activity term: id: GO:0140801 label: histone H2AXY142 kinase activity targets: - preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: Sets the basal pY142 mark, through a kinase domain unrelated to known kinase folds. evidence: - source_id: PMID:19092802 statement: WSTF has intrinsic tyrosine kinase activity and phosphorylates H2AX Tyr142. supporting_text: We show that WSTF has intrinsic tyrosine kinase activity by means of a domain that shares no sequence homology to any known kinase fold. We show that WSTF phosphorylates Tyr 142 of H2A.X - id: eya1_y142_phosphatase label: EYA1 histone H2AX Tyr142 phosphatase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EYA1 term: id: UniProtKB:Q99502 label: Protein phosphatase EYA1 function: preferred_term: histone H2AXY142 phosphatase activity term: id: GO:0140793 label: histone H2AXY142 phosphatase activity targets: - preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Damage-dependent removal of pY142 favours repair over apoptosis; EYA1 and EYA3 are partly redundant. evidence: - source_id: PMID:19234442 statement: EYA dephosphorylates H2AX Tyr142 in response to damage, favouring repair over apoptosis. supporting_text: Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). - id: eya3_y142_phosphatase label: EYA3 histone H2AX Tyr142 phosphatase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EYA3 term: id: UniProtKB:Q99504 label: Protein phosphatase EYA3 function: preferred_term: histone H2AXY142 phosphatase activity term: id: GO:0140793 label: histone H2AXY142 phosphatase activity targets: - preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: EYA paralog with the same H2AX pY142 phosphatase activity (IDA in human GOA). evidence: - source_id: PMID:19234442 statement: The pY142 state determines whether repair or pro-apoptotic factors bind gammaH2AX. supporting_text: This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) - order: 5 role: RNF8-UBE2N-RNF168 chromatin ubiquitylation cascade node: id: dsb_ubiquitin_cascade label: RNF8 and RNF168 ubiquitylation of DSB-flanking chromatin with UBE2N module_type: REGULATORY_STEP description: >- RNF8 docks on ATM-phosphorylated MDC1 via its FHA domain and with UBE2N (UBC13) builds K63-linked ubiquitin chains; RNF168 binds these conjugates and monoubiquitylates nucleosomal H2A/H2AX on Lys13/15, and the RNF168 marks are extended by K63 chains. RNF8 and RNF168 also ubiquitylate KDM4A for proteasomal degradation, unmasking H4K20me2. concepts: - preferred_term: protein K63-linked ubiquitination term: id: GO:0070534 label: protein K63-linked ubiquitination annotons: - id: rnf8_e3 label: RNF8 ubiquitin protein ligase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RNF8 term: id: UniProtKB:O76064 label: E3 ubiquitin-protein ligase RNF8 function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity processes: - preferred_term: protein K63-linked ubiquitination term: id: GO:0070534 label: protein K63-linked ubiquitination locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Initiating E3; recruited by phospho-MDC1. It is inactive on nucleosomal H2A itself, so its chains prime RNF168 recruitment rather than writing the H2A mark. evidence: - source_id: PMID:18001824 statement: RNF8 assembles at DSBs through its FHA domain binding phospho-MDC1 and is needed for 53BP1 and BRCA1 retention. supporting_text: We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. - source_id: PMID:22980979 statement: RNF8 does not ubiquitylate nucleosomal H2A; RNF168 does, on K13-15. supporting_text: RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. gocam_associations: - model: gomodel:65a1f4f800001127 activity: gomodel:65a1f4f800001127/65a1f4f800001191 title: RNF8 ubiquitin protein ligase activity (GO:0061630) - id: ube2n_e2 label: UBE2N (UBC13) ubiquitin conjugating enzyme activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: UBE2N (UBC13) term: id: UniProtKB:P61088 label: Ubiquitin-conjugating enzyme E2 N function: preferred_term: ubiquitin conjugating enzyme activity term: id: GO:0061631 label: ubiquitin conjugating enzyme activity processes: - preferred_term: protein K63-linked ubiquitination term: id: GO:0070534 label: protein K63-linked ubiquitination locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: K63-chain-specific E2 for RNF8 and RNF168; the target of OTUB1 inhibition. evidence: - source_id: PMID:19203578 statement: RNF168 acts with UBC13 to amplify RNF8-dependent K63-linked ubiquitylation. supporting_text: RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. - id: rnf168_e3 label: RNF168 histone H2AK15 ubiquitin ligase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RNF168 term: id: UniProtKB:Q8IYW5 label: E3 ubiquitin-protein ligase RNF168 function: preferred_term: histone H2AK15 ubiquitin ligase activity term: id: GO:0140858 label: histone H2AK15 ubiquitin ligase activity targets: - preferred_term: H2AX term: id: UniProtKB:P16104 label: Histone H2AX - preferred_term: KDM4A (ubiquitylated for degradation, with RNF8) term: id: UniProtKB:O75164 label: Lysine-specific demethylase 4A processes: - preferred_term: protein K63-linked ubiquitination term: id: GO:0070534 label: protein K63-linked ubiquitination locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Rate-limiting amplifier that writes H2AK13/K15ub, the mark read by 53BP1; mutated in RIDDLE syndrome. evidence: - source_id: PMID:19203579 statement: RNF168 amplifies K63-linked conjugates at DSBs to the threshold needed for 53BP1 and BRCA1 retention. supporting_text: by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. - source_id: PMID:22373579 statement: RNF8 and RNF168 ubiquitylate KDM4A, whose degradation frees H4K20me2 for 53BP1. supporting_text: We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. gocam_associations: - model: gomodel:617b481400000460 activity: gomodel:617b481400000460/617b481400000468 title: RNF168 histone H2AK15 ubiquitin ligase activity (GO:0140858), directly provides input for TP53BP1 - order: 6 role: unmasking of H4K20me2 for 53BP1 node: id: h4k20me2_unmasking label: VCP/p97 extraction of L3MBTL1 from H4K20me2 chromatin module_type: REGULATORY_STEP description: >- The H4K20me2 mark that 53BP1 reads is constitutive but occluded by other readers. RNF8-dependent ubiquitylation recruits VCP and its cofactor NPL4, whose ATPase activity releases L3MBTL1 from chromatin; KDM4A is removed in parallel by RNF8/RNF168-dependent proteasomal degradation. annotons: - id: vcp_l3mbtl1_extraction label: VCP ATPase extraction of L3MBTL1 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: VCP (p97) term: id: UniProtKB:P55072 label: Transitional endoplasmic reticulum ATPase function: preferred_term: ATP hydrolysis activity term: id: GO:0016887 label: ATP hydrolysis activity targets: - preferred_term: L3MBTL1 term: id: UniProtKB:Q9Y468 label: Lethal(3)malignant brain tumor-like protein 1 processes: - preferred_term: negative regulation of protein localization to chromatin term: id: GO:0120186 label: negative regulation of protein localization to chromatin locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Segregase that removes the competing H4K20me2 reader L3MBTL1; one of the DNA-repair roles in the VCP review's core functions. evidence: - source_id: PMID:22120668 statement: VCP ATPase activity releases L3MBTL1, which also binds H4K20me2, facilitating 53BP1 recruitment. supporting_text: The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. - order: 7 role: SUMO conjugation and SUMO-targeted ubiquitylation at DSBs node: id: dsb_sumo_arm label: PIAS1/PIAS4 SUMOylation and RNF4 SUMO-targeted ubiquitylation module_type: REGULATORY_STEP description: >- PIAS1 and PIAS4 are recruited to DSBs through their SAP domains and are needed for SUMO accumulation, for productive accrual of 53BP1, BRCA1 and RNF168, and for RNF8/RNF168/BRCA1 ubiquitin-adduct formation. RNF4 recognises SUMOylated MDC1 and ubiquitylates it, turning over MDC1 at damage sites. concepts: - preferred_term: protein sumoylation term: id: GO:0016925 label: protein sumoylation annotons: - id: pias4_sumo_ligase label: PIAS4 SUMO ligase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PIAS4 (PIASy) term: id: UniProtKB:Q8N2W9 label: E3 SUMO-protein ligase PIAS4 function: preferred_term: SUMO ligase activity term: id: GO:0061665 label: SUMO ligase activity processes: - preferred_term: protein sumoylation term: id: GO:0016925 label: protein sumoylation locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Main SUMO1 E3 at DSBs; needed for 53BP1 accumulation. evidence: - source_id: PMID:20016603 statement: PIAS1 and PIAS4 are needed for SUMO accrual and productive 53BP1, BRCA1 and RNF168 association with DSBs. supporting_text: We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. gocam_associations: - model: gomodel:617b481400000460 activity: gomodel:617b481400000460/617b481400000770 title: PIAS4 SUMO ligase activity (GO:0061665), positive regulation of DSB repair via HR - id: pias1_sumo_ligase label: PIAS1 SUMO ligase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PIAS1 term: id: UniProtKB:O75925 label: E3 SUMO-protein ligase PIAS1 function: preferred_term: SUMO ligase activity term: id: GO:0061665 label: SUMO ligase activity processes: - preferred_term: protein sumoylation term: id: GO:0016925 label: protein sumoylation locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: SUMO2/3 E3 at DSBs, needed for BRCA1 accumulation; also SUMOylates MRE11. evidence: - source_id: PMID:20016603 statement: PIAS1 and PIAS4 are required for ubiquitin-adduct formation by RNF8, RNF168 and BRCA1. supporting_text: Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. - source_id: PMID:20016594 statement: PIAS-dependent SUMOylation of BRCA1-BARD1 increases its ubiquitin ligase activity. supporting_text: In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL). gocam_associations: - model: gomodel:65a1f4f800002665 activity: gomodel:65a1f4f800002665/65a1f4f800002731 title: PIAS1 SUMO ligase activity (GO:0061665), SUMOylation of MRE11 - id: rnf4_stubl label: RNF4 SUMO-targeted ubiquitin ligase activity on SUMO-MDC1 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RNF4 term: id: UniProtKB:P78317 label: E3 ubiquitin-protein ligase RNF4 function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity targets: - preferred_term: SUMOylated MDC1 term: id: UniProtKB:Q14676 label: Mediator of DNA damage checkpoint protein 1 locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Recruited by its SUMO-interaction motifs; ubiquitylates SUMO-MDC1 for turnover and is needed for clearance of 53BP1, RNF8 and RNF168 from foci and for efficient resection. evidence: - source_id: PMID:22661230 statement: SUMOylated MDC1 recruits RNF4, which ubiquitylates at the damage site. supporting_text: As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. - source_id: PMID:22661229 statement: RNF4 recruitment needs PIAS1 and PIAS4, and RNF4 regulates MDC1 turnover at damage sites. supporting_text: We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites - order: 8 role: H2B monoubiquitylation and chromatin relaxation (parallel ATM output) node: id: h2b_monoubiquitylation label: ATM-dependent RNF20-RNF40 monoubiquitylation of histone H2B module_type: REGULATORY_STEP description: >- The RNF20-RNF40 heterodimer is recruited to DSBs and phosphorylated by ATM; it monoubiquitylates H2B independently of H2AX, which relaxes chromatin and is needed for timely recruitment of both NHEJ and HR factors. annotons: - id: rnf20_h2b_e3 label: RNF20 histone H2B ubiquitin ligase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RNF20 (BRE1A) term: id: UniProtKB:Q5VTR2 label: E3 ubiquitin-protein ligase BRE1A function: preferred_term: histone H2B C-terminal K residue ubiquitin ligase activity term: id: GO:0140850 label: histone H2B C-terminal K residue ubiquitin ligase activity processes: - preferred_term: DNA repair-dependent chromatin remodeling term: id: GO:0140861 label: DNA repair-dependent chromatin remodeling locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Catalytic RING subunit of the H2B ligase; an ATM substrate. evidence: - source_id: PMID:21362549 statement: DSB-induced H2B monoubiquitylation needs recruitment and ATM-dependent phosphorylation of RNF20-RNF40. supporting_text: "We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer." - id: rnf40_h2b_e3 label: RNF40 ubiquitin ligase partner of RNF20 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RNF40 (BRE1B) term: id: UniProtKB:O75150 label: E3 ubiquitin-protein ligase BRE1B function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity processes: - preferred_term: DNA repair-dependent chromatin remodeling term: id: GO:0140861 label: DNA repair-dependent chromatin remodeling locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Obligate heterodimer partner of RNF20; also an ATM substrate. evidence: - source_id: PMID:21362549 statement: H2B monoubiquitylation is needed for timely recruitment of NHEJ and HR factors. supporting_text: H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. - order: 9 role: repair pathway choice at the ubiquitylated chromatin node: id: pathway_choice label: 53BP1-RIF1 versus BRCA1-A recruitment (repair pathway-choice node) module_type: REGULATORY_STEP description: >- The ubiquitylated, H4K20me2-unmasked chromatin recruits two antagonistic reader systems. 53BP1-RIF1 blocks resection and favours NHEJ, chiefly in G1; BRCA1 (recruited in the BRCA1-A complex, and with CtIP) displaces 53BP1-RIF1 from S phase onward, and KAT5 acetylation of H2AK15 blocks the 53BP1-binding ubiquitin mark, together favouring resection and HR. evidence: - source_id: PMID:23333306 statement: 53BP1 inhibits BRCA1 accumulation in G1, and RIF1 accumulation is antagonised by BRCA1 and CtIP. supporting_text: Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. variant_sets: - id: dsb_reader_choice label: 53BP1 (NHEJ-favouring) versus BRCA1 (HR-favouring) reader systems axis: cell-cycle-dependent repair pathway choice selection: ONE_OR_MORE notes: >- Both readers are recruited by the same RNF168-dependent chromatin marks and compete; which dominates depends mainly on cell-cycle phase (53BP1-RIF1 in G1, BRCA1-CtIP from S phase). They can co-occupy foci, with 53BP1 moved to the periphery by BRCA1, so ONE_OR_MORE rather than EXACTLY_ONE. variants: - id: tp53bp1_rif1_arm label: 53BP1-RIF1 arm (blocks resection, favours NHEJ) module_type: REGULATORY_STEP annotons: - id: tp53bp1_ub_reader label: 53BP1 ubiquitin-modified histone reader (H2AK15ub) participant: selector_type: GENE_PRODUCT gene_product: preferred_term: TP53BP1 (53BP1) term: id: UniProtKB:Q12888 label: TP53-binding protein 1 function: preferred_term: ubiquitin-modified histone reader activity term: id: GO:0061649 label: ubiquitin-modified histone reader activity processes: - preferred_term: protein localization to site of double-strand break term: id: GO:1990166 label: protein localization to site of double-strand break locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: The UDR motif reads H2AK15ub written by RNF168. evidence: - source_id: PMID:23760478 statement: 53BP1 is a bivalent reader of H4K20me2 and H2AK15ub on the same nucleosome. supporting_text: We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. gocam_associations: - model: gomodel:617b481400000460 activity: gomodel:617b481400000460/617b481400000511 title: TP53BP1 ubiquitin-modified histone reader activity (GO:0061649) - id: tp53bp1_h4k20me2_reader label: 53BP1 histone H4K20me2 reader (tandem Tudor) participant: selector_type: GENE_PRODUCT gene_product: preferred_term: TP53BP1 (53BP1) term: id: UniProtKB:Q12888 label: TP53-binding protein 1 function: preferred_term: histone H4K20me2 reader activity term: id: GO:0140005 label: histone H4K20me2 reader activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: The tandem Tudor domain reads H4K20me2 once L3MBTL1 and KDM4A are removed. evidence: - source_id: PMID:17190600 statement: 53BP1 tandem Tudor domains bind H4K20me2 specifically. supporting_text: both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2). gocam_associations: - model: gomodel:617b481400000460 activity: gomodel:617b481400000460/617b481400000475 title: TP53BP1 histone H4K20me2 reader activity (GO:0140005) - id: rif1_53bp1_effector label: RIF1, effector of ATM-phosphorylated 53BP1 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RIF1 term: id: UniProtKB:Q5UIP0 label: Telomere-associated protein RIF1 processes: - preferred_term: negative regulation of double-strand break repair via homologous recombination term: id: GO:2000042 label: negative regulation of double-strand break repair via homologous recombination locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Recruited by the ATM-phosphorylated N-terminal SQ/TQ domain of 53BP1; blocks end resection. No molecular function is asserted because none is established experimentally (it recruits shieldin and PP1, which are outside the module). evidence: - source_id: PMID:23333305 statement: RIF1 is recruited via phospho-53BP1 and suppresses DSB resection. supporting_text: Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. - source_id: PMID:23333306 statement: ATM-dependent 53BP1 phosphorylation recruits RIF1, the critical 53BP1 effector. supporting_text: ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. - id: brca1_a_arm label: BRCA1-A complex and H2AK15 acetylation arm (displaces 53BP1, favours HR) module_type: REGULATORY_STEP annotons: - id: brca1_a_complex_reader label: BRCA1-A complex recognition of K63-linked ubiquitin at DSBs participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: BRCA1-A complex term: id: GO:0070531 label: BRCA1-A complex active_units: - id: brca1a_uimc1 label: RAP80 (UIMC1) ubiquitin reader role: Tandem UIMs bind K63-linked chains and target the complex to DSBs. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: UIMC1 (RAP80) term: id: UniProtKB:Q96RL1 label: BRCA1-A complex subunit RAP80 function: preferred_term: ubiquitin-modified histone reader activity term: id: GO:0061649 label: ubiquitin-modified histone reader activity - id: brca1a_abraxas1 label: ABRAXAS1 scaffold role: Binds the BRCA1 BRCT repeats through a pSXXF motif and recruits RAP80. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ABRAXAS1 term: id: UniProtKB:Q6UWZ7 label: BRCA1-A complex subunit Abraxas 1 function: preferred_term: polyubiquitin modification-dependent protein binding term: id: GO:0031593 label: polyubiquitin modification-dependent protein binding - id: brca1a_brcc3 label: BRCC3 (BRCC36) K63-specific deubiquitinase role: Catalytic DUB subunit; trims K63 chains at DSBs. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BRCC3 (BRCC36) term: id: UniProtKB:P46736 label: Lys-63-specific deubiquitinase BRCC36 function: preferred_term: K63-linked deubiquitinase activity term: id: GO:0061578 label: K63-linked deubiquitinase activity - id: brca1a_babam2 label: BABAM2 (BRE) adaptor role: Structural adaptor bridging BRCC3 and ABRAXAS1. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BABAM2 (BRE) term: id: UniProtKB:Q9NXR7 label: BRISC and BRCA1-A complex member 2 function: preferred_term: protein-macromolecule adaptor activity term: id: GO:0030674 label: protein-macromolecule adaptor activity - id: brca1a_babam1 label: BABAM1 (MERIT40) adaptor role: Structural adaptor required for complex integrity. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BABAM1 (MERIT40) term: id: UniProtKB:Q9NWV8 label: BRISC and BRCA1-A complex member 1 function: preferred_term: protein-macromolecule adaptor activity term: id: GO:0030674 label: protein-macromolecule adaptor activity - id: brca1a_brca1 label: BRCA1 RING E3 ligase role: E3 ligase with BARD1; antagonises 53BP1-RIF1 and, with CtIP, promotes resection. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BRCA1 term: id: UniProtKB:P38398 label: Breast cancer type 1 susceptibility protein function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity - id: brca1a_bard1 label: BARD1 RING partner of BRCA1 role: Obligate heterodimeric RING partner of BRCA1. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: BARD1 term: id: UniProtKB:Q99728 label: BRCA1-associated RING domain protein 1 function: preferred_term: ubiquitin-modified histone reader activity term: id: GO:0061649 label: ubiquitin-modified histone reader activity processes: - preferred_term: protein localization to site of double-strand break term: id: GO:1990166 label: protein localization to site of double-strand break locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Recognises RNF8/RNF168 K63-linked ubiquitin through RAP80 and brings BRCA1-BARD1 and BRCC3 to the break. The reader function is RAP80's; the complex activity is assigned at that unit. evidence: - source_id: PMID:17525341 statement: RAP80 targets the BRCA1-BARD1 and BRCC36 complex to K6/K63-linked ubiquitin at DSBs. supporting_text: RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. - source_id: PMID:17525340 statement: Abraxas binds the BRCA1 BRCT repeats and recruits RAP80. supporting_text: Abraxas recruits the ubiquitin-interacting motif (UIM)-containing protein RAP80 to BRCA1. - source_id: PMID:17525342 statement: RAP80 recruits BRCA1 to damage sites. supporting_text: RAP80 specifically recruits BRCA1 to DNA damage sites and functions with BRCA1 in G2/M checkpoint control. - id: kat5_h2ak15_acetylation label: KAT5 (TIP60) H2AK15 acetylation blocking 53BP1 binding participant: selector_type: GENE_PRODUCT gene_product: preferred_term: KAT5 (TIP60) term: id: UniProtKB:Q92993 label: Histone acetyltransferase KAT5 function: preferred_term: histone H2A acetyltransferase activity term: id: GO:0043998 label: histone H2A acetyltransferase activity targets: - preferred_term: histone H2A Lys15 description: The same residue that RNF168 ubiquitylates; acetylation and ubiquitylation are mutually exclusive. processes: - preferred_term: double-strand break repair via homologous recombination term: id: GO:0000724 label: double-strand break repair via homologous recombination locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- The NuA4/TIP60 complex competes with 53BP1 for H4K20me2 (via MBTD1) and acetylates H2AK15, blocking its ubiquitylation and so limiting 53BP1 binding. evidence: - source_id: PMID:27153538 statement: TIP60 acetylation of H2AK15 blocks its ubiquitylation by RNF168. supporting_text: Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. gocam_associations: - model: gomodel:617b481400000460 activity: gomodel:617b481400000460/617b481400000461 title: KAT5 histone H2A acetyltransferase activity (GO:0043998), part of DSB repair via HR - order: 10 role: signal attenuation and confinement node: id: signal_attenuation label: Reversal and confinement of ATM phosphorylation and chromatin ubiquitylation module_type: REGULATORY_STEP description: >- Negative regulators that keep the signal local and switch it off after repair: phosphatases acting on ATM and gammaH2AX, deubiquitinases and an E2 inhibitor acting on the RNF8/RNF168 marks, and HECT ligases that keep RNF168 limiting. parts: - order: 1 role: dephosphorylation of ATM and gammaH2AX node: id: phosphatase_reversal label: PP2A, PP4, PP6 and WIP1 (PPM1D) dephosphorylation of ATM and gammaH2AX module_type: REGULATORY_STEP annotons: - id: ppp2ca_atm_phosphatase label: PP2A (PPP2CA) restraint of ATM autophosphorylation participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PPP2CA term: id: UniProtKB:P67775 label: Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform function: preferred_term: protein serine/threonine phosphatase activity term: id: GO:0004722 label: protein serine/threonine phosphatase activity targets: - preferred_term: ATM term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Associates with ATM in undamaged cells and restrains Ser1981 autophosphorylation; dissociates after irradiation. PP2A is also required to resolve gammaH2AX foci. evidence: - source_id: PMID:15510216 statement: PP2A A and C subunits bind ATM in undamaged cells, and IR releases PP2A from ATM. supporting_text: Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. - id: ppm1d_gh2ax_phosphatase label: WIP1 (PPM1D) dephosphorylation of gammaH2AX participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PPM1D (WIP1) term: id: UniProtKB:O15297 label: Protein phosphatase 1D function: preferred_term: protein serine/threonine phosphatase activity term: id: GO:0004722 label: protein serine/threonine phosphatase activity targets: - preferred_term: gammaH2AX term: id: UniProtKB:P16104 label: Histone H2AX - preferred_term: ATM (Ser1981) term: id: UniProtKB:Q13315 label: Serine-protein kinase ATM locations: - preferred_term: chromatin term: id: GO:0000785 label: chromatin role_description: >- p53-induced phosphatase that dephosphorylates gammaH2AX (and ATM) to silence the checkpoint during recovery, a negative feedback from the p53 output. evidence: - source_id: PMID:20101220 statement: Wip1 binds chromatin and directly dephosphorylates gammaH2AX, displacing MDC1 when prematurely active. supporting_text: Moreover, Wip1 directly dephosphorylates gamma-H2AX and cells depleted of Wip1 fail to dephosphorylate gamma-H2AX during checkpoint recovery. Conversely, premature activation of Wip1 leads to displacement of MDC1 from damage foci and prevents activation of the checkpoint. - source_id: PMID:20460517 statement: Wip1 dephosphorylates gammaH2AX in vitro and in vivo. supporting_text: effectively dephosphorylates gamma-H2AX in vitro and in vivo - id: ppp4c_gh2ax_phosphatase label: PP4 (PPP4C) dephosphorylation of gammaH2AX participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PPP4C term: id: UniProtKB:P60510 label: Serine/threonine-protein phosphatase 4 catalytic subunit function: preferred_term: protein serine/threonine phosphatase activity term: id: GO:0004722 label: protein serine/threonine phosphatase activity targets: - preferred_term: gammaH2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: chromatin term: id: GO:0000785 label: chromatin role_description: >- PP4C-PP4R2-PP4R3beta complex; dephosphorylates gammaH2AX within nucleosomes, chiefly the ATR-generated mark at replication-associated breaks. evidence: - source_id: PMID:18614045 statement: PP4 dephosphorylates gammaH2AX within mononucleosomes in vitro. supporting_text: PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity - id: ppp6c_gh2ax_phosphatase label: PP6 (PPP6C) dephosphorylation of gammaH2AX participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PPP6C term: id: UniProtKB:O00743 label: Serine/threonine-protein phosphatase 6 catalytic subunit function: preferred_term: protein serine/threonine phosphatase activity term: id: GO:0004722 label: protein serine/threonine phosphatase activity targets: - preferred_term: gammaH2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: chromatin term: id: GO:0000785 label: chromatin role_description: Recruited near DSBs; its loss leaves gammaH2AX high after damage. evidence: - source_id: PMID:21451261 statement: PP6c-containing heterotrimers dephosphorylate gammaH2AX, and PP6c is recruited next to DSBs. supporting_text: Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. - order: 2 role: removal and inhibition of chromatin ubiquitylation node: id: ubiquitin_reversal label: OTUB1 inhibition of UBE2N and USP3/USP16/USP44 deubiquitylation of H2A module_type: REGULATORY_STEP annotons: - id: otub1_ube2n_inhibitor label: OTUB1 non-catalytic inhibition of UBE2N participant: selector_type: GENE_PRODUCT gene_product: preferred_term: OTUB1 term: id: UniProtKB:Q96FW1 label: Ubiquitin thioesterase OTUB1 function: preferred_term: ubiquitin-protein transferase inhibitor activity term: id: GO:0055105 label: ubiquitin-protein transferase inhibitor activity targets: - preferred_term: UBE2N (UBC13) term: id: UniProtKB:P61088 label: Ubiquitin-conjugating enzyme E2 N locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: Binds UBC13 and blocks RNF168-dependent polyubiquitination independently of its DUB activity. evidence: - source_id: PMID:20725033 statement: OTUB1 suppresses RNF168-dependent ubiquitination by binding and inhibiting UBC13, independently of catalysis. supporting_text: Surprisingly, we found that OTUB1 suppresses RNF168-dependent poly-ubiquitination independently of its catalytic activity. OTUB1 does so by binding to and inhibiting UBC13 (also known as UBE2N), the cognate E2 enzyme for RNF168. - id: usp3_h2a_dub label: USP3 histone H2A deubiquitinase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: USP3 term: id: UniProtKB:Q9Y6I4 label: Ubiquitin carboxyl-terminal hydrolase 3 function: preferred_term: histone H2A deubiquitinase activity term: id: GO:0140950 label: histone H2A deubiquitinase activity targets: - preferred_term: H2A / H2AX term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: chromatin term: id: GO:0000785 label: chromatin role_description: Chromatin-associated DUB for uH2A/uH2B, needed for full deubiquitination and gammaH2AX dephosphorylation after IR. evidence: - source_id: PMID:17980597 statement: USP3 deubiquitinates H2A and H2B and is required for full resolution of IR-induced uH2A. supporting_text: USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation - id: usp16_h2a_dub label: USP16 histone H2A deubiquitinase activity (H2AK15ub) participant: selector_type: GENE_PRODUCT gene_product: preferred_term: USP16 term: id: UniProtKB:Q9Y5T5 label: Ubiquitin carboxyl-terminal hydrolase 16 function: preferred_term: histone H2A deubiquitinase activity term: id: GO:0140950 label: histone H2A deubiquitinase activity targets: - preferred_term: H2A (K15ub and K119ub) term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: chromatin term: id: GO:0000785 label: chromatin role_description: HERC2-associated DUB that terminates DSB ubiquitin foci after repair. evidence: - source_id: PMID:25305019 statement: USP16 is needed to terminate DSB ubiquitination and can remove H2AK15ub in vitro. supporting_text: Finally, we demonstrated that USP16 could deubiquitinate both H2A Lys-119 and H2A Lys-15 ubiquitination in vitro. - id: usp44_dub label: USP44 deubiquitinase activity opposing RNF168 accrual participant: selector_type: GENE_PRODUCT gene_product: preferred_term: USP44 term: id: UniProtKB:Q9H0E7 label: Ubiquitin carboxyl-terminal hydrolase 44 function: preferred_term: cysteine-type deubiquitinase activity term: id: GO:0004843 label: cysteine-type deubiquitinase activity targets: - preferred_term: ubiquitylated H2A term: id: UniProtKB:P16104 label: Histone H2AX locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Recruited to RNF168 products at DSBs; overexpression blocks RNF168 accrual. Depletion has mild effects, indicating DUB redundancy. evidence: - source_id: PMID:23615962 statement: USP44 is recruited to RNF168 ubiquitylation products and opposes the RNF8/RNF168 pathway. supporting_text: Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. - order: 3 role: limiting RNF168 abundance node: id: rnf168_limitation label: TRIP12 and UBR5 HECT ligases keep RNF168 limiting module_type: REGULATORY_STEP annotons: - id: trip12_rnf168_limiter label: TRIP12 ubiquitin ligase activity on RNF168 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: TRIP12 term: id: UniProtKB:Q14669 label: E3 ubiquitin-protein ligase TRIP12 function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity targets: - preferred_term: RNF168 term: id: UniProtKB:Q8IYW5 label: E3 ubiquitin-protein ligase RNF168 locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: HECT E3 that controls RNF168 accumulation and prevents spreading of chromatin ubiquitylation. evidence: - source_id: PMID:22884692 statement: TRIP12 and UBR5 control RNF168 accumulation; their loss lets ubiquitin conjugates spread. supporting_text: Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. - id: ubr5_rnf168_limiter label: UBR5 ubiquitin ligase activity on RNF168 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: UBR5 term: id: UniProtKB:O95071 label: E3 ubiquitin-protein ligase UBR5 function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity targets: - preferred_term: RNF168 term: id: UniProtKB:Q8IYW5 label: E3 ubiquitin-protein ligase RNF168 locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: Second HECT E3 acting with TRIP12. evidence: - source_id: PMID:22884692 statement: Depletion of TRIP12 and UBR5 leads to supraphysiological RNF168 and hyperaccumulation of 53BP1 and BRCA1. supporting_text: Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. connections: - source: atm_activation target: h2ax_phosphorylation connection_type: POSITIVELY_REGULATES description: Activated ATM phosphorylates H2AX Ser139 in break-flanking chromatin. - source: h2ax_phosphorylation target: mdc1_amplification connection_type: PROVIDES_INPUT_FOR description: gammaH2AX is the epitope read by the MDC1 BRCT repeat. - source: mdc1_amplification target: atm_activation connection_type: POSITIVELY_REGULATES description: >- MDC1 binds ATM and NBN and retains MRN-ATM on flanking chromatin, the positive feedback that spreads H2AX phosphorylation. - source: h2ax_y142_switch target: mdc1_amplification connection_type: POSITIVELY_REGULATES description: EYA-mediated removal of H2AX pY142 permits MDC1 binding to gammaH2AX. - source: mdc1_amplification target: dsb_ubiquitin_cascade connection_type: POSITIVELY_REGULATES description: ATM-phosphorylated MDC1 recruits RNF8 through its FHA domain. - source: dsb_ubiquitin_cascade target: h4k20me2_unmasking connection_type: POSITIVELY_REGULATES description: RNF8-dependent ubiquitylation recruits VCP-NPL4 to extract L3MBTL1. - source: dsb_ubiquitin_cascade target: pathway_choice connection_type: PROVIDES_INPUT_FOR description: >- RNF168-written H2AK15ub and K63 chains are the marks read by 53BP1 and by RAP80 in the BRCA1-A complex (RNF168 directly provides input for TP53BP1 in gomodel:617b481400000460). - source: h4k20me2_unmasking target: pathway_choice connection_type: POSITIVELY_REGULATES description: Unmasked H4K20me2 is the second mark required for 53BP1 binding. - source: dsb_sumo_arm target: dsb_ubiquitin_cascade connection_type: POSITIVELY_REGULATES description: PIAS1/PIAS4 SUMOylation is required for RNF8-, RNF168- and BRCA1-dependent ubiquitin-adduct formation. - source: atm_activation target: h2b_monoubiquitylation connection_type: POSITIVELY_REGULATES description: ATM phosphorylates and activates RNF20-RNF40 at DSBs. - source: signal_attenuation target: atm_activation connection_type: NEGATIVELY_REGULATES description: PP2A (and WIP1) dephosphorylate ATM Ser1981. - source: signal_attenuation target: h2ax_phosphorylation connection_type: NEGATIVELY_REGULATES description: WIP1, PP4, PP6 and PP2A remove gammaH2AX. - source: signal_attenuation target: dsb_ubiquitin_cascade connection_type: NEGATIVELY_REGULATES description: >- OTUB1 inhibits UBE2N, USP3/USP16/USP44 remove H2A ubiquitin, and TRIP12/UBR5 keep RNF168 limiting. knowledge_gaps: - gap_statement: >- How MRN converts DNA end binding into ATM activation is unresolved: whether ATM monomerisation depends on MRN-generated single-stranded oligonucleotides, on DNA end tethering by RAD50, or on chromatin changes sensed through KAT5 binding to H3K9me3, and how these mechanisms are weighted in cells. gap_kind: - BIOLOGY - gap_statement: >- How 53BP1-RIF1 and BRCA1 displace each other at the same ubiquitylated chromatin is incompletely understood, including how much depends on BRCA1-BARD1 ubiquitylation of H2A K125-129, on KAT5 H2AK15 acetylation, on 53BP1 dephosphorylation, or on CtIP-dependent remodelling, and what molecular activity RIF1 itself contributes. gap_kind: - BIOLOGY - gap_statement: >- The relative contributions and substrate selectivity of the many phosphatases (PP2A, PP4, PP6, WIP1) and deubiquitinases (USP3, USP16, USP44, BRCC3, OTUB1) that attenuate the signal are unclear; single depletions have mild effects, suggesting redundancy, and which enzyme terminates the ATM-generated, as opposed to the ATR-generated, gammaH2AX in vivo is not known. gap_kind: - BIOLOGY