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

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.

MODULE:atm_dsb_signalingDRAFTCONCRETESignaling Pathwaymodules/atm_dsb_signaling.yaml
signal transduction in response to DNA damageGO:0042770 protein localization to site of double-strand breakGO:1990166
PW:0001361
ataxia telangiectasia-mutated (ATM) signaling pathway
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).
GO:0042770
signal transduction in response to DNA damage
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.
GO:0000077
DNA damage checkpoint signaling
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.
gomodel:617b481400000460
KAT5 involvement in DNA repair via homologous recombination (Human)
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.
gomodel:65a1f4f800000487
Phosphorylation of H2AX by ATM or ATR promotes recruitment of NBN and MRN complex to DNA damage sites (Human).
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.
PMID:15790808
ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
MRN is the DSB sensor that recruits and activates ATM.
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.
PMID:16427009
MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals.
H2AX, MDC1 and ATM form the positive feedback loop at the core of the module.
At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM.
PMID:23333306
A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.
The module ends at the 53BP1 versus BRCA1 pathway-choice node.
DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1.

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.

16Nodes
13Parts
1Variant Sets
2Variants
34Annotons
13Connections

Derived QC

Recommended-field compliance

30.8% recommended fields populated
  • knowledge_gaps[0] · boundary (0/1)
  • knowledge_gaps[0] · status (0/1)
  • knowledge_gaps[0] · provenance (0/1)
  • knowledge_gaps[1] · boundary (0/1)
  • knowledge_gaps[1] · status (0/1)
  • knowledge_gaps[1] · provenance (0/1)
  • knowledge_gaps[2] · boundary (0/1)
  • knowledge_gaps[2] · status (0/1)
  • knowledge_gaps[2] · provenance (0/1)

Module deep research

✗ none found

No MODULE:atm_dsb_signaling deep-research report alongside the module YAML.

Leaf nodes lacking representative members

✓ every leaf node grounds to a representative protein.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

9 gene-function check(s) reflected in core functions · 0 conflict(s) · 31 evidence or coverage gap(s) (42 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

AnnotonParticipantFunctionAssessment
mrn_atm_activator PROTEIN_COMPLEX
Gene 		protein serine/threonine kinase activator activity
GO:0043539 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
mrn_atm_activator/mrn_mre11 MRE11
UniProtKB:P49959
Complex Unit 		DNA endonuclease activity
GO:0004520 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
mrn_atm_activator/mrn_rad50 RAD50
UniProtKB:Q92878
Complex Unit 		ATP hydrolysis activity
GO:0016887 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
mrn_atm_activator/mrn_nbn NBN
UniProtKB:O60934
Complex Unit 		protein serine/threonine kinase activator activity
GO:0043539 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
kat5_atm_acetylation KAT5 (TIP60)
UniProtKB:Q92993
Gene 		protein-lysine-acetyltransferase activity
GO:0061733 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ppp5c_atm_activation PPP5C
UniProtKB:P53041
Gene 		protein serine/threonine phosphatase activity
GO:0004722 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
atm_kinase ATM
UniProtKB:Q13315
Gene 		protein serine/threonine kinase activity
GO:0004674 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
atm_h2ax_kinase ATM
UniProtKB:Q13315
Gene 		histone H2AXS139 kinase activity
GO:0035979 		Annotation Supported
A retained annotation or curated replacement supports this function, but core_functions does not yet reflect it.
h2ax_scaffold H2AX
UniProtKB:P16104
Gene 		chromatin-protein adaptor activity
GO:0140463 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
mdc1_gh2ax_reader MDC1
UniProtKB:Q14676
Gene 		histone reader activity
GO:0140566 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
baz1b_y142_kinase BAZ1B (WSTF)
UniProtKB:Q9UIG0
Gene 		histone H2AXY142 kinase activity
GO:0140801 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
eya1_y142_phosphatase EYA1
UniProtKB:Q99502
Gene 		histone H2AXY142 phosphatase activity
GO:0140793 		Annotation Supported
A retained annotation or curated replacement supports this function, but core_functions does not yet reflect it.
eya3_y142_phosphatase EYA3
UniProtKB:Q99504
Gene 		histone H2AXY142 phosphatase activity
GO:0140793 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
rnf8_e3 RNF8
UniProtKB:O76064
Gene 		ubiquitin protein ligase activity
GO:0061630 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ube2n_e2 UBE2N (UBC13)
UniProtKB:P61088
Gene 		ubiquitin conjugating enzyme activity
GO:0061631 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
rnf168_e3 RNF168
UniProtKB:Q8IYW5
Gene 		histone H2AK15 ubiquitin ligase activity
GO:0140858 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
vcp_l3mbtl1_extraction VCP (p97)
UniProtKB:P55072
Gene 		ATP hydrolysis activity
GO:0016887 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
pias4_sumo_ligase PIAS4 (PIASy)
UniProtKB:Q8N2W9
Gene 		SUMO ligase activity
GO:0061665 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
pias1_sumo_ligase PIAS1
UniProtKB:O75925
Gene 		SUMO ligase activity
GO:0061665 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rnf4_stubl RNF4
UniProtKB:P78317
Gene 		ubiquitin protein ligase activity
GO:0061630 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
rnf20_h2b_e3 RNF20 (BRE1A)
UniProtKB:Q5VTR2
Gene 		histone H2B C-terminal K residue ubiquitin ligase activity
GO:0140850 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
rnf40_h2b_e3 RNF40 (BRE1B)
UniProtKB:O75150
Gene 		ubiquitin protein ligase activity
GO:0061630 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
tp53bp1_ub_reader TP53BP1 (53BP1)
UniProtKB:Q12888
Gene 		ubiquitin-modified histone reader activity
GO:0061649 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
tp53bp1_h4k20me2_reader TP53BP1 (53BP1)
UniProtKB:Q12888
Gene 		histone H4K20me2 reader activity
GO:0140005 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
brca1_a_complex_reader PROTEIN_COMPLEX
Gene 		ubiquitin-modified histone reader activity
GO:0061649 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
brca1_a_complex_reader/brca1a_uimc1 UIMC1 (RAP80)
UniProtKB:Q96RL1
Complex Unit 		ubiquitin-modified histone reader activity
GO:0061649 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
brca1_a_complex_reader/brca1a_abraxas1 ABRAXAS1
UniProtKB:Q6UWZ7
Complex Unit 		polyubiquitin modification-dependent protein binding
GO:0031593 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
brca1_a_complex_reader/brca1a_brcc3 BRCC3 (BRCC36)
UniProtKB:P46736
Complex Unit 		K63-linked deubiquitinase activity
GO:0061578 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
brca1_a_complex_reader/brca1a_babam2 BABAM2 (BRE)
UniProtKB:Q9NXR7
Complex Unit 		protein-macromolecule adaptor activity
GO:0030674 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
brca1_a_complex_reader/brca1a_babam1 BABAM1 (MERIT40)
UniProtKB:Q9NWV8
Complex Unit 		protein-macromolecule adaptor activity
GO:0030674 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
brca1_a_complex_reader/brca1a_brca1 BRCA1
UniProtKB:P38398
Complex Unit 		ubiquitin protein ligase activity
GO:0061630 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
kat5_h2ak15_acetylation KAT5 (TIP60)
UniProtKB:Q92993
Gene 		histone H2A acetyltransferase activity
GO:0043998 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ppp2ca_atm_phosphatase PPP2CA
UniProtKB:P67775
Gene 		protein serine/threonine phosphatase activity
GO:0004722 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ppm1d_gh2ax_phosphatase PPM1D (WIP1)
UniProtKB:O15297
Gene 		protein serine/threonine phosphatase activity
GO:0004722 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ppp4c_gh2ax_phosphatase PPP4C
UniProtKB:P60510
Gene 		protein serine/threonine phosphatase activity
GO:0004722 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ppp6c_gh2ax_phosphatase PPP6C
UniProtKB:O00743
Gene 		protein serine/threonine phosphatase activity
GO:0004722 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
otub1_ube2n_inhibitor OTUB1
UniProtKB:Q96FW1
Gene 		ubiquitin-protein transferase inhibitor activity
GO:0055105 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
usp3_h2a_dub USP3
UniProtKB:Q9Y6I4
Gene 		histone H2A deubiquitinase activity
GO:0140950 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
usp16_h2a_dub USP16
UniProtKB:Q9Y5T5
Gene 		histone H2A deubiquitinase activity
GO:0140950 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
usp44_dub USP44
UniProtKB:Q9H0E7
Gene 		cysteine-type deubiquitinase activity
GO:0004843 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
trip12_rnf168_limiter TRIP12
UniProtKB:Q14669
Gene 		ubiquitin protein ligase activity
GO:0061630 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
ubr5_rnf168_limiter UBR5
UniProtKB:O95071
Gene 		ubiquitin protein ligase activity
GO:0061630 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.

Feedback loops (advisory, prose-driven: a regulator counts as "feedback" when its own description says so)

✓ every negative regulator described as feedback has an upstream edge closing the loop.

  • signal_attenuation ⊣ atm_activation [input]
    signal_attenuation is a negative regulator with no upstream edge (not described as feedback)

Gene-review completeness (11/41 grounded genes reviewed)

5 complete review(s) · 7 with deep research · 30 missing review · 4 reviewed but lacking deep research

Gene Review Complete Deep research
ABRAXAS1 Q6UWZ7 ✓ 47/50 ✓
ATM Q13315 ✓ 285/325 ✗
BABAM1 Q9NWV8 ✓ ✓ ✓
BABAM2 Q9NXR7 ✓ ✓ ✓
BARD1 Q99728 ✓ 160/162 ✗
BRCA1 P38398 ✓ 249/275 ✓
EYA1 Q99502 ✓ 30/33 ✗
NBN O60934 ✓ 259/260 ✗
PPP6C O00743 ✗ — —
PPM1D (WIP1) O15297 ✗ — —
RNF40 (BRE1B) O75150 ✗ — —
KDM4A (ubiquitylated for degradation, with RNF8) O75164 ✗ — —
RNF8 O76064 ✗ — —
UBR5 O95071 ✗ — —
H2AX P16104 ✗ — —
BRCC3 (BRCC36) P46736 ✗ — —
MRE11 P49959 ✗ — —
PPP5C P53041 ✗ — —
PPP4C P60510 ✗ — —
UBE2N (UBC13) P61088 ✗ — —
RNF4 P78317 ✗ — —
PIAS1 O75925 ✓ ✓ ✓
PPP2CA P67775 ✓ ✓ ✓
TP53BP1 Q12888 ✗ — —
TRIP12 Q14669 ✗ — —
MDC1 Q14676 ✗ — —
RIF1 Q5UIP0 ✗ — —
RNF20 Q5VTR2 ✗ — —
RNF168 Q8IYW5 ✗ — —
PIAS4 (PIASy) Q8N2W9 ✗ — —
RAD50 Q92878 ✗ — —
KAT5 (TIP60) Q92993 ✗ — —
OTUB1 Q96FW1 ✗ — —
UIMC1 (RAP80) Q96RL1 ✗ — —
EYA3 Q99504 ✗ — —
USP44 Q9H0E7 ✗ — —
BAZ1B (WSTF) Q9UIG0 ✗ — —
L3MBTL1 Q9Y468 ✗ — —
USP16 Q9Y5T5 ✗ — —
USP3 Q9Y6I4 ✗ — —
VCP P55072 ✓ ✓ ✓

Details

Context
Homo sapiensNCBITaxon:9606
site of double-strand breakGO:0035861
ATM signaling at DNA double-strand breaksSignaling Pathwayatm_dsb_signaling
signal transduction in response to DNA damageGO:0042770 protein localization to site of double-strand breakGO:1990166
Context
Homo sapiensNCBITaxon:9606
site of double-strand breakGO:0035861

Connections

atm_activation -> h2ax_phosphorylation Positively Regulates
Activated ATM phosphorylates H2AX Ser139 in break-flanking chromatin.
gammaH2AX is the epitope read by the MDC1 BRCT repeat.
mdc1_amplification -> atm_activation Positively Regulates
MDC1 binds ATM and NBN and retains MRN-ATM on flanking chromatin, the positive feedback that spreads H2AX phosphorylation.
h2ax_y142_switch -> mdc1_amplification Positively Regulates
EYA-mediated removal of H2AX pY142 permits MDC1 binding to gammaH2AX.
mdc1_amplification -> dsb_ubiquitin_cascade Positively Regulates
ATM-phosphorylated MDC1 recruits RNF8 through its FHA domain.
dsb_ubiquitin_cascade -> h4k20me2_unmasking Positively Regulates
RNF8-dependent ubiquitylation recruits VCP-NPL4 to extract L3MBTL1.
dsb_ubiquitin_cascade -> pathway_choice Provides Input For
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).
h4k20me2_unmasking -> pathway_choice Positively Regulates
Unmasked H4K20me2 is the second mark required for 53BP1 binding.
dsb_sumo_arm -> dsb_ubiquitin_cascade Positively Regulates
PIAS1/PIAS4 SUMOylation is required for RNF8-, RNF168- and BRCA1-dependent ubiquitin-adduct formation.
atm_activation -> h2b_monoubiquitylation Positively Regulates
ATM phosphorylates and activates RNF20-RNF40 at DSBs.
signal_attenuation -> atm_activation Negatively Regulates
PP2A (and WIP1) dephosphorylate ATM Ser1981.
signal_attenuation -> h2ax_phosphorylation Negatively Regulates
WIP1, PP4, PP6 and PP2A remove gammaH2AX.
signal_attenuation -> dsb_ubiquitin_cascade Negatively Regulates
OTUB1 inhibits UBE2N, USP3/USP16/USP44 remove H2A ubiquitin, and TRIP12/UBR5 keep RNF168 limiting.
Part 1: DSB sensing by MRN and ATM activation
MRN recruitment and activation of ATM, with KAT5 acetylation of ATMRegulatory Stepatm_activation

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

MRN complex activates ATM at DNA ends
mrn_atm_activator
Participant: Protein Complex: MRE11-RAD50-NBN (MRN) complex
Protein Complex:
MRE11-RAD50-NBN (MRN) complexGO:0030870
Active units:
MRE11 nuclease subunit
Participant: Gene Product: MRE11
Gene Product:
MRE11UniProtKB:P49959
Role: Binds and processes DNA ends; nuclease core of the complex.
Function:
DNA endonuclease activityGO:0004520
RAD50 ATPase subunit
Participant: Gene Product: RAD50
Gene Product:
RAD50UniProtKB:Q92878
Role: ATP-dependent DNA end binding and tethering; DNA unwinding by MRN is needed for ATM stimulation.
Function:
ATP hydrolysis activityGO:0016887
NBN (NBS1) ATM-recruiting subunit
Participant: Gene Product: NBN
Gene Product:
NBNUniProtKB:O60934
Role: Its conserved C-terminal motif binds and recruits ATM to the break.
Function:
protein serine/threonine kinase activator activityGO:0043539
PMID:15758953
The NBN C-terminal motif is required for ATM recruitment to damage and for ATM signalling.
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

protein serine/threonine kinase activator activityGO:0043539
Targets: ATMUniProtKB:Q13315

Locations

site of double-strand breakGO:0035861

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.

PMID:15790808
MRN monomerises and activates ATM in vitro; DNA unwinding by MRN is essential.
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.
PMID:14532133
Functional MRN is required for ATM activation in cells.
Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways.
KAT5 (TIP60) acetylation of ATM
kat5_atm_acetylation
Participant: Gene Product: KAT5 (TIP60)
Gene Product:
KAT5 (TIP60)UniProtKB:Q92993

Function

protein-lysine-acetyltransferase activityGO:0061733
Targets: ATMUniProtKB:Q13315

Locations

site of double-strand breakGO:0035861

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.

PMID:16141325
DNA damage induces Tip60-dependent ATM acetylation, and Tip60 loss blocks ATM activation.
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.
PP5 (PPP5C) requirement for ATM activation
ppp5c_atm_activation
Participant: Gene Product: PPP5C
Gene Product:
PPP5CUniProtKB:P53041

Function

protein serine/threonine phosphatase activityGO:0004722

Locations

nucleusGO:0005634

Binds ATM in a damage-inducible manner; catalytically inactive PP5 blocks ATM Ser1981 autophosphorylation. Its phosphatase substrate in this role is not known.

PMID:14871926
Catalytic activity of PP5 is needed for ATM autophosphorylation and checkpoint signalling.
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.
ATM serine/threonine kinase activity on DSB-response substrates
atm_kinase
Participant: Gene Product: ATM
Gene Product:
ATMUniProtKB:Q13315

Function

protein serine/threonine kinase activityGO:0004674
Targets: ATM (Ser1981 autophosphorylation)UniProtKB:Q13315 NBNUniProtKB:O60934 MDC1UniProtKB:Q14676 TP53BP1UniProtKB:Q12888 BRCA1UniProtKB:P38398 RNF20UniProtKB:Q5VTR2

Processes

DNA damage checkpoint signalingGO:0000077

Locations

site of double-strand breakGO:0035861

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.

PMID:12556884
Intermolecular Ser1981 autophosphorylation dissociates inactive ATM dimers.
Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity.
Part 2: H2AX Ser139 phosphorylation and gammaH2AX scaffold
ATM phosphorylation of H2AX Ser139 (gammaH2AX)Reactionh2ax_phosphorylation

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

ATM histone H2AX Ser139 kinase activity
atm_h2ax_kinase
Participant: Gene Product: ATM
Gene Product:
ATMUniProtKB:Q13315

Function

histone H2AXS139 kinase activityGO:0035979
Targets: H2AXUniProtKB:P16104

Locations

site of double-strand breakGO:0035861

The major H2AX Ser139 kinase after DSBs.

PMID:11571274
ATM phosphorylates H2AX in vitro and is required for H2AX phosphorylation and foci in cells.
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.
gammaH2AX chromatin-protein adaptor (MDC1 docking site)
h2ax_scaffold
Participant: Gene Product: H2AX
Gene Product:
H2AXUniProtKB:P16104

Function

chromatin-protein adaptor activityGO:0140463

Processes

protein localization to site of double-strand breakGO:1990166

Locations

site of double-strand breakGO:0035861

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).

PMID:16377563
gammaH2AX phosphorylation is the main recruitment signal, read directly by MDC1.
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.
Part 3: MDC1 reading of gammaH2AX and feed-forward amplification
MDC1 binding to gammaH2AX and accumulation of MRN-ATMRegulatory Stepmdc1_amplification

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

MDC1 histone reader activity (gammaH2AX)
mdc1_gh2ax_reader
Participant: Gene Product: MDC1
Gene Product:
MDC1UniProtKB:Q14676

Function

histone reader activityGO:0140566
Targets: gammaH2AX (H2AX phospho-Ser139)UniProtKB:P16104

Processes

protein localization to site of double-strand breakGO:1990166

Locations

site of double-strand breakGO:0035861

Mediator and signal amplifier; Mdc1-null mice recapitulate many H2AX-null phenotypes.

PMID:16377563
The MDC1 BRCT repeat binds the gammaH2AX phosphoepitope directly.
These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage.
PMID:16427009
MDC1 binds gammaH2AX via its BRCT domain and ATM via its FHA domain, accumulating active ATM.
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.
Part 4: H2AX Tyr142 phosphorylation switch gating MDC1 binding
BAZ1B (WSTF) phosphorylation and EYA1/EYA3 dephosphorylation of H2AX Tyr142Regulatory Steph2ax_y142_switch

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

BAZ1B (WSTF) histone H2AX Tyr142 kinase activity
baz1b_y142_kinase
Participant: Gene Product: BAZ1B (WSTF)
Gene Product:
BAZ1B (WSTF)UniProtKB:Q9UIG0

Function

histone H2AXY142 kinase activityGO:0140801
Targets: H2AXUniProtKB:P16104

Locations

nucleusGO:0005634

Sets the basal pY142 mark, through a kinase domain unrelated to known kinase folds.

PMID:19092802
WSTF has intrinsic tyrosine kinase activity and phosphorylates H2AX Tyr142.
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
EYA1 histone H2AX Tyr142 phosphatase activity
eya1_y142_phosphatase
Participant: Gene Product: EYA1
Gene Product:
EYA1UniProtKB:Q99502

Function

histone H2AXY142 phosphatase activityGO:0140793
Targets: H2AXUniProtKB:P16104

Locations

nucleusGO:0005634

Damage-dependent removal of pY142 favours repair over apoptosis; EYA1 and EYA3 are partly redundant.

PMID:19234442
EYA dephosphorylates H2AX Tyr142 in response to damage, favouring repair over apoptosis.
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).
EYA3 histone H2AX Tyr142 phosphatase activity
eya3_y142_phosphatase
Participant: Gene Product: EYA3
Gene Product:
EYA3UniProtKB:Q99504

Function

histone H2AXY142 phosphatase activityGO:0140793
Targets: H2AXUniProtKB:P16104

Locations

nucleusGO:0005634

EYA paralog with the same H2AX pY142 phosphatase activity (IDA in human GOA).

PMID:19234442
The pY142 state determines whether repair or pro-apoptotic factors bind gammaH2AX.
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)
Part 5: RNF8-UBE2N-RNF168 chromatin ubiquitylation cascade
RNF8 and RNF168 ubiquitylation of DSB-flanking chromatin with UBE2NRegulatory Stepdsb_ubiquitin_cascade

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.

protein K63-linked ubiquitinationGO:0070534

Annotons

RNF8 ubiquitin protein ligase activity
rnf8_e3
Participant: Gene Product: RNF8
Gene Product:
RNF8UniProtKB:O76064

Function

ubiquitin protein ligase activityGO:0061630

Processes

protein K63-linked ubiquitinationGO:0070534

Locations

site of double-strand breakGO:0035861

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.

PMID:18001824
RNF8 assembles at DSBs through its FHA domain binding phospho-MDC1 and is needed for 53BP1 and BRCA1 retention.
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.
PMID:22980979
RNF8 does not ubiquitylate nucleosomal H2A; RNF168 does, on K13-15.
RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15.
UBE2N (UBC13) ubiquitin conjugating enzyme activity
ube2n_e2
Participant: Gene Product: UBE2N (UBC13)
Gene Product:
UBE2N (UBC13)UniProtKB:P61088

Function

ubiquitin conjugating enzyme activityGO:0061631

Processes

protein K63-linked ubiquitinationGO:0070534

Locations

site of double-strand breakGO:0035861

K63-chain-specific E2 for RNF8 and RNF168; the target of OTUB1 inhibition.

PMID:19203578
RNF168 acts with UBC13 to amplify RNF8-dependent K63-linked ubiquitylation.
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.
RNF168 histone H2AK15 ubiquitin ligase activity
rnf168_e3
Participant: Gene Product: RNF168
Gene Product:
RNF168UniProtKB:Q8IYW5

Function

histone H2AK15 ubiquitin ligase activityGO:0140858
Targets: H2AXUniProtKB:P16104 KDM4A (ubiquitylated for degradation, with RNF8)UniProtKB:O75164

Processes

protein K63-linked ubiquitinationGO:0070534

Locations

site of double-strand breakGO:0035861

Rate-limiting amplifier that writes H2AK13/K15ub, the mark read by 53BP1; mutated in RIDDLE syndrome.

PMID:19203579
RNF168 amplifies K63-linked conjugates at DSBs to the threshold needed for 53BP1 and BRCA1 retention.
by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1.
PMID:22373579
RNF8 and RNF168 ubiquitylate KDM4A, whose degradation frees H4K20me2 for 53BP1.
We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168.
Part 6: unmasking of H4K20me2 for 53BP1
VCP/p97 extraction of L3MBTL1 from H4K20me2 chromatinRegulatory Steph4k20me2_unmasking

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

VCP ATPase extraction of L3MBTL1
vcp_l3mbtl1_extraction
Participant: Gene Product: VCP (p97)
Gene Product:
VCP (p97)UniProtKB:P55072

Function

ATP hydrolysis activityGO:0016887
Targets: L3MBTL1UniProtKB:Q9Y468

Processes

negative regulation of protein localization to chromatinGO:0120186

Locations

site of double-strand breakGO:0035861

Segregase that removes the competing H4K20me2 reader L3MBTL1; one of the DNA-repair roles in the VCP review's core functions.

PMID:22120668
VCP ATPase activity releases L3MBTL1, which also binds H4K20me2, facilitating 53BP1 recruitment.
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.
Part 7: SUMO conjugation and SUMO-targeted ubiquitylation at DSBs
PIAS1/PIAS4 SUMOylation and RNF4 SUMO-targeted ubiquitylationRegulatory Stepdsb_sumo_arm

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.

protein sumoylationGO:0016925

Annotons

PIAS4 SUMO ligase activity
pias4_sumo_ligase
Participant: Gene Product: PIAS4 (PIASy)
Gene Product:
PIAS4 (PIASy)UniProtKB:Q8N2W9

Function

SUMO ligase activityGO:0061665

Processes

protein sumoylationGO:0016925

Locations

site of double-strand breakGO:0035861

Main SUMO1 E3 at DSBs; needed for 53BP1 accumulation.

PMID:20016603
PIAS1 and PIAS4 are needed for SUMO accrual and productive 53BP1, BRCA1 and RNF168 association with DSBs.
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.
PIAS1 SUMO ligase activity
pias1_sumo_ligase
Participant: Gene Product: PIAS1
Gene Product:
PIAS1UniProtKB:O75925

Function

SUMO ligase activityGO:0061665

Processes

protein sumoylationGO:0016925

Locations

site of double-strand breakGO:0035861

SUMO2/3 E3 at DSBs, needed for BRCA1 accumulation; also SUMOylates MRE11.

PMID:20016603
PIAS1 and PIAS4 are required for ubiquitin-adduct formation by RNF8, RNF168 and BRCA1.
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.
PMID:20016594
PIAS-dependent SUMOylation of BRCA1-BARD1 increases its ubiquitin ligase activity.
In vitro SUMO modification of the BRCA1/BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO-regulated ubiquitin ligase (SRUbL).
RNF4 SUMO-targeted ubiquitin ligase activity on SUMO-MDC1
rnf4_stubl
Participant: Gene Product: RNF4
Gene Product:
RNF4UniProtKB:P78317

Function

ubiquitin protein ligase activityGO:0061630
Targets: SUMOylated MDC1UniProtKB:Q14676

Locations

site of double-strand breakGO:0035861

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.

PMID:22661230
SUMOylated MDC1 recruits RNF4, which ubiquitylates at the damage site.
As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site.
PMID:22661229
RNF4 recruitment needs PIAS1 and PIAS4, and RNF4 regulates MDC1 turnover at damage sites.
We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites
Part 8: H2B monoubiquitylation and chromatin relaxation (parallel ATM output)
ATM-dependent RNF20-RNF40 monoubiquitylation of histone H2BRegulatory Steph2b_monoubiquitylation

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

RNF20 histone H2B ubiquitin ligase activity
rnf20_h2b_e3
Participant: Gene Product: RNF20 (BRE1A)
Gene Product:
RNF20 (BRE1A)UniProtKB:Q5VTR2

Function

histone H2B C-terminal K residue ubiquitin ligase activityGO:0140850

Processes

DNA repair-dependent chromatin remodelingGO:0140861

Locations

site of double-strand breakGO:0035861

Catalytic RING subunit of the H2B ligase; an ATM substrate.

PMID:21362549
DSB-induced H2B monoubiquitylation needs recruitment and ATM-dependent phosphorylation of RNF20-RNF40.
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.
RNF40 ubiquitin ligase partner of RNF20
rnf40_h2b_e3
Participant: Gene Product: RNF40 (BRE1B)
Gene Product:
RNF40 (BRE1B)UniProtKB:O75150

Function

ubiquitin protein ligase activityGO:0061630

Processes

DNA repair-dependent chromatin remodelingGO:0140861

Locations

site of double-strand breakGO:0035861

Obligate heterodimer partner of RNF20; also an ATM substrate.

PMID:21362549
H2B monoubiquitylation is needed for timely recruitment of NHEJ and HR factors.
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.
Part 9: repair pathway choice at the ubiquitylated chromatin
53BP1-RIF1 versus BRCA1-A recruitment (repair pathway-choice node)Regulatory Steppathway_choice

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.

PMID:23333306
53BP1 inhibits BRCA1 accumulation in G1, and RIF1 accumulation is antagonised by BRCA1 and CtIP.
Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP.
Variant set: 53BP1 (NHEJ-favouring) versus BRCA1 (HR-favouring) reader systems by cell-cycle-dependent repair pathway choice (One Or More)

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.

53BP1-RIF1 arm (blocks resection, favours NHEJ)Regulatory Steptp53bp1_rif1_arm

Annotons

53BP1 ubiquitin-modified histone reader (H2AK15ub)
tp53bp1_ub_reader
Participant: Gene Product: TP53BP1 (53BP1)
Gene Product:
TP53BP1 (53BP1)UniProtKB:Q12888

Function

ubiquitin-modified histone reader activityGO:0061649

Processes

protein localization to site of double-strand breakGO:1990166

Locations

site of double-strand breakGO:0035861

The UDR motif reads H2AK15ub written by RNF168.

PMID:23760478
53BP1 is a bivalent reader of H4K20me2 and H2AK15ub on the same nucleosome.
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.
53BP1 histone H4K20me2 reader (tandem Tudor)
tp53bp1_h4k20me2_reader
Participant: Gene Product: TP53BP1 (53BP1)
Gene Product:
TP53BP1 (53BP1)UniProtKB:Q12888

Function

histone H4K20me2 reader activityGO:0140005

Locations

site of double-strand breakGO:0035861

The tandem Tudor domain reads H4K20me2 once L3MBTL1 and KDM4A are removed.

PMID:17190600
53BP1 tandem Tudor domains bind H4K20me2 specifically.
both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2).
RIF1, effector of ATM-phosphorylated 53BP1
rif1_53bp1_effector
Participant: Gene Product: RIF1
Gene Product:
RIF1UniProtKB:Q5UIP0

Processes

negative regulation of double-strand break repair via homologous recombinationGO:2000042

Locations

site of double-strand breakGO:0035861

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).

PMID:23333305
RIF1 is recruited via phospho-53BP1 and suppresses DSB resection.
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.
PMID:23333306
ATM-dependent 53BP1 phosphorylation recruits RIF1, the critical 53BP1 effector.
ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair.
BRCA1-A complex and H2AK15 acetylation arm (displaces 53BP1, favours HR)Regulatory Stepbrca1_a_arm

Annotons

BRCA1-A complex recognition of K63-linked ubiquitin at DSBs
brca1_a_complex_reader
Participant: Protein Complex: BRCA1-A complex
Protein Complex:
BRCA1-A complexGO:0070531
Active units:
RAP80 (UIMC1) ubiquitin reader
Participant: Gene Product: UIMC1 (RAP80)
Gene Product:
UIMC1 (RAP80)UniProtKB:Q96RL1
Role: Tandem UIMs bind K63-linked chains and target the complex to DSBs.
Function:
ubiquitin-modified histone reader activityGO:0061649
ABRAXAS1 scaffold
Participant: Gene Product: ABRAXAS1
Gene Product:
ABRAXAS1UniProtKB:Q6UWZ7
Role: Binds the BRCA1 BRCT repeats through a pSXXF motif and recruits RAP80.
Function:
polyubiquitin modification-dependent protein bindingGO:0031593
BRCC3 (BRCC36) K63-specific deubiquitinase
Participant: Gene Product: BRCC3 (BRCC36)
Gene Product:
BRCC3 (BRCC36)UniProtKB:P46736
Role: Catalytic DUB subunit; trims K63 chains at DSBs.
Function:
K63-linked deubiquitinase activityGO:0061578
BABAM2 (BRE) adaptor
Participant: Gene Product: BABAM2 (BRE)
Gene Product:
BABAM2 (BRE)UniProtKB:Q9NXR7
Role: Structural adaptor bridging BRCC3 and ABRAXAS1.
Function:
protein-macromolecule adaptor activityGO:0030674
BABAM1 (MERIT40) adaptor
Participant: Gene Product: BABAM1 (MERIT40)
Gene Product:
BABAM1 (MERIT40)UniProtKB:Q9NWV8
Role: Structural adaptor required for complex integrity.
Function:
protein-macromolecule adaptor activityGO:0030674
BRCA1 RING E3 ligase
Participant: Gene Product: BRCA1
Gene Product:
BRCA1UniProtKB:P38398
Role: E3 ligase with BARD1; antagonises 53BP1-RIF1 and, with CtIP, promotes resection.
Function:
ubiquitin protein ligase activityGO:0061630
BARD1 RING partner of BRCA1
Participant: Gene Product: BARD1
Gene Product:
BARD1UniProtKB:Q99728
Role: Obligate heterodimeric RING partner of BRCA1.

Function

ubiquitin-modified histone reader activityGO:0061649

Processes

protein localization to site of double-strand breakGO:1990166

Locations

site of double-strand breakGO:0035861

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.

PMID:17525341
RAP80 targets the BRCA1-BARD1 and BRCC36 complex to K6/K63-linked ubiquitin at DSBs.
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.
PMID:17525340
Abraxas binds the BRCA1 BRCT repeats and recruits RAP80.
Abraxas recruits the ubiquitin-interacting motif (UIM)-containing protein RAP80 to BRCA1.
PMID:17525342
RAP80 recruits BRCA1 to damage sites.
RAP80 specifically recruits BRCA1 to DNA damage sites and functions with BRCA1 in G2/M checkpoint control.
KAT5 (TIP60) H2AK15 acetylation blocking 53BP1 binding
kat5_h2ak15_acetylation
Participant: Gene Product: KAT5 (TIP60)
Gene Product:
KAT5 (TIP60)UniProtKB:Q92993

Function

histone H2A acetyltransferase activityGO:0043998
Targets: histone H2A Lys15

Processes

double-strand break repair via homologous recombinationGO:0000724

Locations

site of double-strand breakGO:0035861

The NuA4/TIP60 complex competes with 53BP1 for H4K20me2 (via MBTD1) and acetylates H2AK15, blocking its ubiquitylation and so limiting 53BP1 binding.

PMID:27153538
TIP60 acetylation of H2AK15 blocks its ubiquitylation by RNF168.
Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation.
Part 10: signal attenuation and confinement
Reversal and confinement of ATM phosphorylation and chromatin ubiquitylationRegulatory Stepsignal_attenuation

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.

Part 1: dephosphorylation of ATM and gammaH2AX
PP2A, PP4, PP6 and WIP1 (PPM1D) dephosphorylation of ATM and gammaH2AXRegulatory Stepphosphatase_reversal

Annotons

PP2A (PPP2CA) restraint of ATM autophosphorylation
ppp2ca_atm_phosphatase
Participant: Gene Product: PPP2CA
Gene Product:
PPP2CAUniProtKB:P67775

Function

protein serine/threonine phosphatase activityGO:0004722
Targets: ATMUniProtKB:Q13315

Locations

nucleusGO:0005634

Associates with ATM in undamaged cells and restrains Ser1981 autophosphorylation; dissociates after irradiation. PP2A is also required to resolve gammaH2AX foci.

PMID:15510216
PP2A A and C subunits bind ATM in undamaged cells, and IR releases PP2A from ATM.
Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity.
WIP1 (PPM1D) dephosphorylation of gammaH2AX
ppm1d_gh2ax_phosphatase
Participant: Gene Product: PPM1D (WIP1)
Gene Product:
PPM1D (WIP1)UniProtKB:O15297

Function

protein serine/threonine phosphatase activityGO:0004722
Targets: gammaH2AXUniProtKB:P16104 ATM (Ser1981)UniProtKB:Q13315

Locations

chromatinGO:0000785

p53-induced phosphatase that dephosphorylates gammaH2AX (and ATM) to silence the checkpoint during recovery, a negative feedback from the p53 output.

PMID:20101220
Wip1 binds chromatin and directly dephosphorylates gammaH2AX, displacing MDC1 when prematurely active.
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.
PMID:20460517
Wip1 dephosphorylates gammaH2AX in vitro and in vivo.
effectively dephosphorylates gamma-H2AX in vitro and in vivo
PP4 (PPP4C) dephosphorylation of gammaH2AX
ppp4c_gh2ax_phosphatase
Participant: Gene Product: PPP4C
Gene Product:
PPP4CUniProtKB:P60510

Function

protein serine/threonine phosphatase activityGO:0004722
Targets: gammaH2AXUniProtKB:P16104

Locations

chromatinGO:0000785

PP4C-PP4R2-PP4R3beta complex; dephosphorylates gammaH2AX within nucleosomes, chiefly the ATR-generated mark at replication-associated breaks.

PMID:18614045
PP4 dephosphorylates gammaH2AX within mononucleosomes in vitro.
PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity
PP6 (PPP6C) dephosphorylation of gammaH2AX
ppp6c_gh2ax_phosphatase
Participant: Gene Product: PPP6C
Gene Product:
PPP6CUniProtKB:O00743

Function

protein serine/threonine phosphatase activityGO:0004722
Targets: gammaH2AXUniProtKB:P16104

Locations

chromatinGO:0000785

Recruited near DSBs; its loss leaves gammaH2AX high after damage.

PMID:21451261
PP6c-containing heterotrimers dephosphorylate gammaH2AX, and PP6c is recruited next to DSBs.
Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR.
Part 2: removal and inhibition of chromatin ubiquitylation
OTUB1 inhibition of UBE2N and USP3/USP16/USP44 deubiquitylation of H2ARegulatory Stepubiquitin_reversal

Annotons

OTUB1 non-catalytic inhibition of UBE2N
otub1_ube2n_inhibitor
Participant: Gene Product: OTUB1
Gene Product:
OTUB1UniProtKB:Q96FW1

Function

ubiquitin-protein transferase inhibitor activityGO:0055105
Targets: UBE2N (UBC13)UniProtKB:P61088

Locations

nucleusGO:0005634

Binds UBC13 and blocks RNF168-dependent polyubiquitination independently of its DUB activity.

PMID:20725033
OTUB1 suppresses RNF168-dependent ubiquitination by binding and inhibiting UBC13, independently of catalysis.
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.
USP3 histone H2A deubiquitinase activity
usp3_h2a_dub
Participant: Gene Product: USP3
Gene Product:
USP3UniProtKB:Q9Y6I4

Function

histone H2A deubiquitinase activityGO:0140950
Targets: H2A / H2AXUniProtKB:P16104

Locations

chromatinGO:0000785

Chromatin-associated DUB for uH2A/uH2B, needed for full deubiquitination and gammaH2AX dephosphorylation after IR.

PMID:17980597
USP3 deubiquitinates H2A and H2B and is required for full resolution of IR-induced uH2A.
USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation
USP16 histone H2A deubiquitinase activity (H2AK15ub)
usp16_h2a_dub
Participant: Gene Product: USP16
Gene Product:
USP16UniProtKB:Q9Y5T5

Function

histone H2A deubiquitinase activityGO:0140950
Targets: H2A (K15ub and K119ub)UniProtKB:P16104

Locations

chromatinGO:0000785

HERC2-associated DUB that terminates DSB ubiquitin foci after repair.

PMID:25305019
USP16 is needed to terminate DSB ubiquitination and can remove H2AK15ub in vitro.
Finally, we demonstrated that USP16 could deubiquitinate both H2A Lys-119 and H2A Lys-15 ubiquitination in vitro.
USP44 deubiquitinase activity opposing RNF168 accrual
usp44_dub
Participant: Gene Product: USP44
Gene Product:
USP44UniProtKB:Q9H0E7

Function

cysteine-type deubiquitinase activityGO:0004843
Targets: ubiquitylated H2AUniProtKB:P16104

Locations

site of double-strand breakGO:0035861

Recruited to RNF168 products at DSBs; overexpression blocks RNF168 accrual. Depletion has mild effects, indicating DUB redundancy.

PMID:23615962
USP44 is recruited to RNF168 ubiquitylation products and opposes the RNF8/RNF168 pathway.
Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites.
Part 3: limiting RNF168 abundance
TRIP12 and UBR5 HECT ligases keep RNF168 limitingRegulatory Steprnf168_limitation

Annotons

TRIP12 ubiquitin ligase activity on RNF168
trip12_rnf168_limiter
Participant: Gene Product: TRIP12
Gene Product:
TRIP12UniProtKB:Q14669

Function

ubiquitin protein ligase activityGO:0061630
Targets: RNF168UniProtKB:Q8IYW5

Locations

nucleusGO:0005634

HECT E3 that controls RNF168 accumulation and prevents spreading of chromatin ubiquitylation.

PMID:22884692
TRIP12 and UBR5 control RNF168 accumulation; their loss lets ubiquitin conjugates spread.
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.
UBR5 ubiquitin ligase activity on RNF168
ubr5_rnf168_limiter
Participant: Gene Product: UBR5
Gene Product:
UBR5UniProtKB:O95071

Function

ubiquitin protein ligase activityGO:0061630
Targets: RNF168UniProtKB:Q8IYW5

Locations

nucleusGO:0005634

Second HECT E3 acting with TRIP12.

PMID:22884692
Depletion of TRIP12 and UBR5 leads to supraphysiological RNF168 and hyperaccumulation of 53BP1 and BRCA1.
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.