id: MODULE:nonhomologous_end_joining title: Classical non-homologous end joining of DNA double-strand breaks — Ku70/Ku80 end binding, DNA-PKcs recruitment and autophosphorylation, Artemis/PNKP/APTX/APLF end processing, pol mu/pol lambda/TdT gap filling, and XRCC4-LIG4 ligation with XLF and PAXX; XRCC6/XRCC5/PRKDC/DCLRE1C/PNKP/APTX/APLF/POLM/POLL/DNTT/XRCC4/LIG4/NHEJ1/PAXX description: >- Classical non-homologous end joining (c-NHEJ) rejoins the two ends of a DNA double-strand break (DSB) directly, without a homologous template. It operates throughout the cell cycle, is the predominant DSB repair pathway in G1 and in non-dividing cells, and is the joining step of V(D)J recombination and class switch recombination in lymphocytes. The Ku heterodimer (Ku70/XRCC6 and Ku80/XRCC5) threads onto each broken end through a preformed ring and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs, PRKDC) to form the DNA-PK holoenzyme, which tethers the two ends in a synaptic complex. DNA-PKcs is activated by the DNA end, autophosphorylates (which remodels the synapse and releases the ends for processing) and phosphorylates other NHEJ factors, including the nuclease Artemis (DCLRE1C). Incompatible or damaged ends are processed by enzymes chosen by end chemistry: the Artemis-DNA-PKcs complex opens hairpins and trims 5' and 3' overhangs; the polynucleotide kinase-phosphatase PNKP restores 5'-phosphate and 3'-hydroxyl termini; aprataxin (APTX) removes 5'-adenylate left by abortive ligation; and APLF, recruited by poly(ADP-ribose) and by Ku, acts as a scaffold that retains the ligase complex and has reported nuclease activity. Gaps are filled by the family X polymerases pol mu (POLM) and pol lambda (POLL), and in developing lymphocytes the template-independent terminal deoxynucleotidyl transferase (TdT, DNTT) adds non-templated nucleotides to V(D)J coding ends. The ends are finally sealed by DNA ligase IV (LIG4) in a constitutive complex with its scaffold XRCC4; the XRCC4 paralogs XLF (NHEJ1) and PAXX bind XRCC4 and Ku respectively, bridge and align the ends, and stimulate ligation, with XLF and PAXX partly redundant. Upstream, the 53BP1-RIF1 axis of ATM signalling protects DSB ends from resection in G1 and so favours c-NHEJ over homologous recombination. status: DRAFT scope: CONCRETE evidence: - source_id: PW:0000203 title: non-homologous end joining pathway of double-strand break repair statement: >- The module boundary follows the RGD Pathway Ontology term and its diagram. The diagram draws Xrcc5 and Xrcc6 positively affecting and associated with Prkdc, Prkdc associated with and positively affecting Dclre1c (Artemis), the "Pol X members" associated with Xrcc5 and Xrcc6, Lig4, Nhej1, Pnkp, Aptx and Aplf associated with Xrcc4, and Rif1 associated with Tp53bp1; the ATM signalling pathway (PW:0001361) is the upstream diagram link. Derived through projects/RGD_PATHWAY_ONTOLOGY (pathways/PW_0000203.yaml). - source_id: GO:0006303 title: double-strand break repair via nonhomologous end joining statement: >- Mapped to PW:0000203 (skos:relatedMatch, from the PW definition cross-reference) in projects/RGD_PATHWAY_ONTOLOGY/data/module_seeds.yaml. Its two GO children separate classical from alternative end joining; this module models only the classical branch, so its process concept is the child GO:0097680. - source_id: PMID:25941166 title: XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair. statement: The seven core NHEJ proteins define the core of this module; PAXX (XLS) is an eighth. supporting_text: "Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV)." - source_id: PMID:8422676 title: "The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen." statement: DNA-PK is a two-component kinase, Ku plus a ~350 kDa catalytic subunit, activated by DNA ends. supporting_text: "DNA cross-linking and coimmunoprecipitation studies indicate that the catalytic 350 kd DNA-PK component is directed to DNA by protein-protein interactions with Ku." notes: >- Derived from the RGD Pathway Ontology diagram PW:0000203 through the RGD_PATHWAY_ONTOLOGY project's diagram->module route (seed in projects/RGD_PATHWAY_ONTOLOGY/data/module_seeds.yaml, tier related_module_only; the only related module found by the seed was bacterial_nonhomologous_end_joining). RGD curates 16 human genes to PW:0000203: APLF APTX DCLRE1C DNTT LIG4 NHEJ1 PNKP POLL POLM PRKDC RBBP8 RIF1 TP53BP1 XRCC4 XRCC5 XRCC6. Thirteen are modelled as annotons here; TP53BP1 and RIF1 are included by module_ref (part 1), and RBBP8 is excluded (decision 3). One gene product not on the RGD list is added: PAXX, the XRCC4/XLF paralog identified in 2015, after the RGD diagram was drawn. Molecular functions were taken from each protein's experimental GO annotations in QuickGO (November 2026) and, where a gene review exists (DCLRE1C, PRKDC), checked against its core functions. Boundary decisions. (1) Upstream signalling is not re-modelled. Part 1 is a module_ref to atm_dsb_signaling#tp53bp1_rif1_arm, the 53BP1-RIF1 arm of that module's pathway-choice node, which already carries TP53BP1 and RIF1 with their activities and GO-CAM links (gomodel:617b481400000460, the seed's best-overlap GO-CAM for PW:0000203, whose only overlap with this pathway is TP53BP1; it is not linked again here). PW:0001361, which RGD links from the NHEJ diagram, is mentioned here but not listed as an evidence source. The part is optional because Ku-dependent joining does not itself require 53BP1. Shieldin and CST, the downstream effectors of RIF1, are not on the RGD list and are not modelled. (2) Alternative end joining is excluded and recorded as context only. RGD's gene list decides scope, and none of the alt-EJ machinery (POLQ, PARP1, LIG3 with XRCC1, LIG1) is curated to PW:0000203; the RGD description and diagram describe only the Ku-DNA-PK-XRCC4-LIG4 pathway. Alt-EJ is mechanistically distinct (it needs resection and microhomology, is Ku-independent and is suppressed by Ku), has its own GO term (GO:0097681), and would be a separate module. For the same reason the module concept is GO:0097680 (classical NHEJ), the child of the mapped term GO:0006303. (3) RBBP8 (CtIP) is excluded although RGD curates it to this pathway: its activity is the initiation of end resection, which commits a break away from c-NHEJ toward HR or alt-EJ; it is a participant of modules/homologous_recombination_dsb_repair.yaml. Its presence on the RGD list probably reflects its role in pathway choice. (4) End processing is a ROUTE variant set of distinct activities for distinct end chemistries (ONE_OR_MORE) in an optional part. PNKP has two annotons, one per catalytic activity. APLF is given DNA endonuclease activity, its experimental IDA row (PMID:17396150) and the activity RGD states, but its best-documented NHEJ role is as a poly(ADP-ribose)- and Ku-binding scaffold that retains XRCC4-LIG4 (PMID:21211721), and no cited study shows that the nuclease is needed for joining in cells; this is recorded as a knowledge gap rather than resolved. APTX activity was demonstrated on single-strand nicks and gaps; its DSB role rests on its XRCC4 interaction. (5) The Pol X polymerases are a REALIZER variant set in an optional part. DNTT (TdT) is on the RGD list and is included as a lymphocyte-restricted variant because its N-addition is part of NHEJ-mediated V(D)J coding-joint formation; V(D)J recombination itself (RAG1/RAG2) is not modelled. (6) Ligation is one part with the XRCC4-LIG4 complex and the XRCC4 paralogs XLF and PAXX as separate annotons. XLF carries DNA end binding (IDA) and PAXX the generic molecular adaptor activity (IDA), the only non-binding experimental MF rows each has; XRCC4 carries protein-macromolecule adaptor activity (IMP). (7) DNA-PKcs is modelled as a single annoton with DNA-dependent protein kinase activity and its two functionally established targets (itself and Artemis); its many other substrates, its H2AX kinase role (backup to ATM, see atm_dsb_signaling) and its non-repair roles are outside the module. (8) Ku is modelled with DNA end binding; Ku70's 5'-dRP/AP lyase activity is noted only in a role. (9) No substrate is modelled as a participant; every annoton is an enzyme or a DNA-binding scaffold. (10) modules/bacterial_nonhomologous_end_joining.yaml is the bacterial analogue (Ku with the multifunctional LigD) and is not referenced; it shares the Ku end-binding step but not the eukaryotic DNA-PK, XRCC4-LIG4 or Pol X machinery. GO-CAM and GOA observations. No human GO-CAM in gocams/index.tsv models the core c-NHEJ machinery (Ku, DNA-PKcs, Artemis, Pol X, XRCC4-LIG4, XLF, PAXX) in GO:0006303 or GO:0097680; the activities that are in that process in the index are 53BP1, RNF168, RAP80 and TOPBP1, which belong to the ATM module. The cached models that contain members do so for other processes (XRCC4 in gomodel:63c0ac2b00002148, DSB attachment to the nuclear envelope; APLF in gomodel:627d95ee00002067, PARP-dependent recruitment; PRKDC in cGAS and TONSL-MMS22L models), so no gocam_associations are asserted. POLM has no experimental biological-process row in human GOA, and DCLRE1C has no experimental process row for NHEJ or V(D)J recombination although both are well established. PANTHER family ids and PTNs are deliberately not asserted. module: id: nonhomologous_end_joining label: Classical non-homologous end joining of DNA double-strand breaks module_type: BIOLOGICAL_PROCESS concepts: - preferred_term: double-strand break repair via classical nonhomologous end joining term: id: GO:0097680 label: double-strand break repair via classical nonhomologous end joining description: >- The classical (Ku- and LIG4-dependent) child of GO:0006303, the term mapped to PW:0000203. Alternative end joining (GO:0097681) is outside the module. 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: upstream end protection by the 53BP1-RIF1 axis optional: true node: id: nhej_upstream_signalling label: 53BP1-RIF1 restraint of end resection (by reference) description: >- Included by reference to the NHEJ-favouring 53BP1-RIF1 arm of the pathway-choice node of the ATM DSB signalling module, which models how ATM-phosphorylated 53BP1 recruits RIF1 to block resection, chiefly in G1. TP53BP1 and RIF1 are on the RGD gene list for PW:0000203. Marked optional because Ku-dependent joining itself does not require 53BP1. module_ref: atm_dsb_signaling#tp53bp1_rif1_arm - order: 2 role: DSB end recognition by Ku node: id: ku_end_binding label: Ku70/Ku80 heterodimer binding to the broken DNA ends module_type: REGULATORY_STEP description: >- Ku threads onto each double-stranded end within seconds and acts as the toolbelt that recruits DNA-PKcs, XRCC4-LIG4, XLF, PAXX, APLF and the Pol X polymerases. annotons: - id: ku_dna_end_binding label: Ku70:Ku80 DNA end binding participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: Ku heterodimer term: id: GO:0043564 label: Ku70:Ku80 complex active_units: - id: ku_xrcc6 label: Ku70 (XRCC6) role: Ring-forming subunit; also carries 5'-dRP/AP lyase activity that cleans abasic sites near ends. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: XRCC6 (Ku70) term: id: UniProtKB:P12956 label: DNA repair protein Ku70 - id: ku_xrcc5 label: Ku80 (XRCC5) role: Ring-forming subunit whose C-terminal motif recruits DNA-PKcs. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: XRCC5 (Ku80) term: id: UniProtKB:P13010 label: DNA repair protein Ku80 function: preferred_term: DNA end binding term: id: GO:0045027 label: DNA end binding function: preferred_term: DNA end binding term: id: GO:0045027 label: DNA end binding locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- End binding requires the heterodimer (both subunits carry contributes_to rows for GO:0045027 in GOA; XRCC5 also an enables row). evidence: - source_id: PMID:11493912 statement: Ku forms a preformed ring that encircles duplex DNA at the break and supports the ends for processing and ligation. supporting_text: "Ku70 and Ku80 share a common topology and form a dyad-symmetrical molecule with a preformed ring that encircles duplex DNA." - order: 3 role: DNA-PK holoenzyme assembly, synapsis and kinase activation node: id: dna_pk_activation label: DNA-PKcs recruitment by Ku and autophosphorylation module_type: REGULATORY_STEP description: >- Ku recruits DNA-PKcs to form the DNA-PK holoenzyme, which bridges the two ends in a long-range synaptic complex. DNA end binding activates the kinase; autophosphorylation in trans remodels the complex and permits end processing and ligation, and DNA-PKcs phosphorylates and activates Artemis. annotons: - id: dna_pkcs_kinase label: DNA-PKcs DNA-dependent protein kinase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PRKDC (DNA-PKcs) term: id: UniProtKB:P78527 label: DNA-dependent protein kinase catalytic subunit function: preferred_term: DNA-dependent protein kinase activity term: id: GO:0004677 label: DNA-dependent protein kinase activity targets: - preferred_term: PRKDC (autophosphorylation) term: id: UniProtKB:P78527 label: DNA-dependent protein kinase catalytic subunit - preferred_term: DCLRE1C (Artemis) term: id: UniProtKB:Q96SD1 label: Protein artemis locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- The pathway kinase; the PRKDC gene review lists GO:0004677 as core. DNA-PKcs also phosphorylates XRCC4, LIG4 and H2AX, which are not asserted as targets here because the functional consequence for joining is not established for each. evidence: - source_id: PMID:8422676 statement: DNA-PK binds to and is activated by DNA ends, and is Ku plus a ~350 kDa catalytic subunit. supporting_text: "Interestingly, we find that the DNA-PK binds to and is activated by the ends of DNA molecules." - source_id: PMID:11955432 statement: DNA-PKcs phosphorylates Artemis on complex formation. supporting_text: "Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on 5' and 3' overhangs, as well as hairpins." - order: 4 role: processing of incompatible or damaged DNA ends optional: true node: id: end_processing label: End processing by Artemis, PNKP, aprataxin and APLF module_type: REACTION description: >- Ends that cannot be ligated directly are processed by enzymes selected by end structure. Each is recruited through Ku, DNA-PKcs or XRCC4, and any subset may act on a given break, in any order. The part is optional because blunt or compatible ends with ligatable termini need no processing. variant_sets: - id: end_processing_enzymes label: End-processing activities chosen by end chemistry axis: DNA end structure variant_kind: ROUTE selection: ONE_OR_MORE notes: >- These are not alternatives for one chemistry but different activities for different lesions (overhangs and hairpins; 3'-phosphate and 5'-hydroxyl; 5'-adenylate). ONE_OR_MORE because a single break can need several. variants: - id: artemis_processing label: Artemis-DNA-PKcs nuclease (overhang trimming, hairpin opening) module_type: REACTION annotons: - id: artemis_nuclease label: Artemis single-stranded DNA endonuclease activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: DCLRE1C (Artemis) term: id: UniProtKB:Q96SD1 label: Protein artemis function: preferred_term: single-stranded DNA endonuclease activity term: id: GO:0000014 label: single-stranded DNA endonuclease activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Alone a 5'-3' exonuclease; in complex with and phosphorylated by DNA-PKcs it cleaves 5' and 3' overhangs and opens RAG-generated coding-end hairpins (essential for V(D)J recombination). The DCLRE1C gene review lists GO:0000014 as core. evidence: - source_id: PMID:11955432 statement: The Artemis-DNA-PKcs complex opens RAG hairpins and processes overhangs. supporting_text: "Finally, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex." - id: pnkp_processing label: PNKP restoration of 5'-phosphate and 3'-hydroxyl termini module_type: REACTION annotons: - id: pnkp_kinase label: PNKP DNA 5'-kinase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PNKP term: id: UniProtKB:Q96T60 label: Bifunctional polynucleotide phosphatase/kinase function: preferred_term: ATP-dependent polydeoxyribonucleotide 5'-hydroxyl-kinase activity term: id: GO:0046404 label: ATP-dependent polydeoxyribonucleotide 5'-hydroxyl-kinase activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Phosphorylates 5'-hydroxyl termini; recruited through its FHA domain to CK2-phosphorylated XRCC4. evidence: - source_id: PMID:10446193 statement: Human PNKP phosphorylates 5'-hydroxyl termini and dephosphorylates 3'-phosphate termini. supporting_text: "Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5'-hydroxyl termini and dephosphorylate 3'-phosphate termini" - source_id: PMID:15385968 statement: PNKP acts in NHEJ through an FHA-dependent interaction with phosphorylated XRCC4. supporting_text: "We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4." - id: pnkp_phosphatase label: PNKP DNA 3'-phosphatase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PNKP term: id: UniProtKB:Q96T60 label: Bifunctional polynucleotide phosphatase/kinase function: preferred_term: polynucleotide 3'-phosphatase activity term: id: GO:0046403 label: polynucleotide 3'-phosphatase activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Removes 3'-phosphate groups to give ligatable 3'-hydroxyl ends. evidence: - source_id: PMID:10446193 statement: Recombinant PNKP has both kinase and phosphatase activities. supporting_text: "the recombinant protein was shown to possess the kinase and phosphatase activities" - id: aptx_processing label: Aprataxin removal of 5'-adenylate from abortive ligation module_type: REACTION annotons: - id: aptx_deadenylase label: Aprataxin DNA 5'-adenosine monophosphate hydrolase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: APTX term: id: UniProtKB:Q7Z2E3 label: Aprataxin function: preferred_term: DNA 5'-adenosine monophosphate hydrolase activity term: id: GO:0033699 label: DNA 5'-adenosine monophosphate hydrolase activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Resolves abortive ligation intermediates; binds XRCC4 (and XRCC1 in single-strand break repair) through its FHA domain. Its activity was shown on nicks and gaps, and its contribution at DSBs is inferred from the XRCC4 interaction. evidence: - source_id: PMID:16964241 statement: Aprataxin releases adenylate from 5'-phosphate termini, giving ligatable ends. supporting_text: "Specifically, aprataxin catalyses the nucleophilic release of adenylate groups covalently linked to 5'-phosphate termini at single-strand nicks and gaps, resulting in the production of 5'-phosphate termini that can be efficiently rejoined." - id: aplf_processing label: APLF scaffold and nuclease at DSB ends module_type: REACTION annotons: - id: aplf_nuclease_scaffold label: APLF DNA endonuclease activity at protruding ends participant: selector_type: GENE_PRODUCT gene_product: preferred_term: APLF term: id: UniProtKB:Q8IW19 label: Aprataxin and PNK-like factor function: preferred_term: DNA endonuclease activity term: id: GO:0004520 label: DNA endonuclease activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Recruited by poly(ADP-ribose) (PARP3) and by Ku; retains XRCC4-LIG4 on chromatin to accelerate ligation. RGD and the original report assign it endo/exonuclease activity, which is asserted here, but its main documented NHEJ role is as a scaffold (see notes). evidence: - source_id: PMID:17396150 statement: APLF binds Ku86, LIG4 and phosphorylated XRCC4 and has nuclease activity at protruding ends. supporting_text: "PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends." - source_id: PMID:21211721 statement: APLF promotes retention of XRCC4-LIG4 in chromatin and accelerates NHEJ with PARP3. supporting_text: "We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ)." - order: 5 role: gap filling by family X polymerases optional: true node: id: gap_filling label: Pol X gap filling and nucleotide addition at aligned ends module_type: REACTION description: >- Pol mu and pol lambda, recruited to Ku-bound ends through their BRCT domains, fill gaps between partially complementary overhangs; TdT adds non-templated nucleotides to coding ends in developing lymphocytes. The part is optional because ends that align without gaps need no synthesis. concepts: - preferred_term: gap filling involved in double-strand break repair via nonhomologous end joining term: id: GO:0061674 label: gap filling involved in double-strand break repair via nonhomologous end joining variant_sets: - id: pol_x_polymerases label: Pol mu, pol lambda or TdT axis: template dependence and cell type variant_kind: REALIZER selection: ONE_OR_MORE notes: >- A gradient of template dependence (PMID:16061182): pol lambda is the most template-dependent, pol mu can synthesise across ends lacking complementary template, and TdT is template-independent and is expressed only in early lymphoid cells. variants: - id: pol_mu_gap_filling label: Pol mu (POLM) module_type: REACTION annotons: - id: polm_polymerase label: Pol mu DNA-directed DNA polymerase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLM term: id: UniProtKB:Q9NP87 label: DNA-directed DNA/RNA polymerase mu function: preferred_term: DNA-directed DNA polymerase activity term: id: GO:0003887 label: DNA-directed DNA polymerase activity processes: - preferred_term: gap filling involved in double-strand break repair via nonhomologous end joining term: id: GO:0061674 label: gap filling involved in double-strand break repair via nonhomologous end joining locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Can extend primers that lack complementary template, instructed by the other DNA end. evidence: - source_id: PMID:16061182 statement: Pol mu and pol lambda are recruited by NHEJ factors to fill gaps between partially complementary ends. supporting_text: "We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ." - id: pol_lambda_gap_filling label: Pol lambda (POLL) module_type: REACTION annotons: - id: poll_polymerase label: Pol lambda DNA-directed DNA polymerase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLL term: id: UniProtKB:Q9UGP5 label: DNA polymerase lambda function: preferred_term: DNA-directed DNA polymerase activity term: id: GO:0003887 label: DNA-directed DNA polymerase activity processes: - preferred_term: gap filling involved in double-strand break repair via nonhomologous end joining term: id: GO:0061674 label: gap filling involved in double-strand break repair via nonhomologous end joining locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: Template-dependent gap filling; stimulated and recruited by XRCC4, XLF and PAXX. evidence: - source_id: PMID:16061182 statement: Pol mu and pol lambda are recruited by NHEJ factors to fill gaps between partially complementary ends. supporting_text: "We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ." - id: tdt_n_addition label: TdT (DNTT), lymphoid V(D)J recombination module_type: REACTION context: cell_types: - preferred_term: lymphocyte term: id: CL:0000542 label: lymphocyte annotons: - id: dntt_nucleotidyltransferase label: TdT DNA nucleotidylexotransferase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: DNTT (TdT) term: id: UniProtKB:P04053 label: DNA nucleotidylexotransferase function: preferred_term: DNA nucleotidylexotransferase activity term: id: GO:0003912 label: DNA nucleotidylexotransferase activity locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Adds N nucleotides to V(D)J coding ends; contributes junctional diversity rather than repair fidelity, and is restricted to early B and T cells. evidence: - source_id: PMID:16061182 statement: TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates. supporting_text: "Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear." - order: 6 role: end bridging and ligation node: id: ligation label: XRCC4-LIG4 ligation with XLF and PAXX module_type: REACTION description: >- LIG4, stabilised and stimulated by XRCC4, seals the aligned ends. XLF binds XRCC4 and forms filaments that bridge the ends; PAXX binds Ku and promotes assembly of the ligation complex. XLF and PAXX are partly redundant. concepts: - preferred_term: double-strand break repair via classical nonhomologous end joining term: id: GO:0097680 label: double-strand break repair via classical nonhomologous end joining annotons: - id: xrcc4_lig4_ligase label: XRCC4-LIG4 DNA ligase (ATP) activity participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: XRCC4-DNA ligase IV complex term: id: GO:0032807 label: DNA ligase IV complex active_units: - id: lig4_ligase label: LIG4 catalytic subunit role: ATP-dependent DNA ligase that seals the break. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: LIG4 term: id: UniProtKB:P49917 label: DNA ligase 4 function: preferred_term: DNA ligase (ATP) activity term: id: GO:0003910 label: DNA ligase (ATP) activity - id: lig4_xrcc4 label: XRCC4 scaffold role: Stabilises and stimulates LIG4 and docks PNKP, APTX, APLF and XLF. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: XRCC4 term: id: UniProtKB:Q13426 label: DNA repair protein XRCC4 function: preferred_term: protein-macromolecule adaptor activity term: id: GO:0030674 label: protein-macromolecule adaptor activity function: preferred_term: DNA ligase (ATP) activity term: id: GO:0003910 label: DNA ligase (ATP) activity processes: - preferred_term: double-strand break repair via classical nonhomologous end joining term: id: GO:0097680 label: double-strand break repair via classical nonhomologous end joining locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- The ligase specific to c-NHEJ; LIG4 deficiency causes LIG4 syndrome with immunodeficiency and radiosensitivity. evidence: - source_id: PMID:9242410 statement: XRCC4 binds LIG4 and stimulates its ligation of DSBs. supporting_text: "Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV." - id: xlf_bridging label: XLF (NHEJ1) end bridging with XRCC4 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: NHEJ1 (XLF, Cernunnos) term: id: UniProtKB:Q9H9Q4 label: Non-homologous end-joining factor 1 function: preferred_term: DNA end binding term: id: GO:0045027 label: DNA end binding processes: - preferred_term: double-strand break repair via classical nonhomologous end joining term: id: GO:0097680 label: double-strand break repair via classical nonhomologous end joining locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- XRCC4 paralog that binds the XRCC4-LIG4 complex and DNA; XLF-XRCC4 filaments bridge the ends. Mutated in NHEJ deficiency with microcephaly and immunodeficiency. evidence: - source_id: PMID:16439205 statement: XLF binds XRCC4-LIG4 and is required for NHEJ. supporting_text: "We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ." - id: paxx_adaptor label: PAXX Ku-binding adaptor promoting ligation participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PAXX term: id: UniProtKB:Q9BUH6 label: Protein PAXX function: preferred_term: molecular adaptor activity term: id: GO:0060090 label: molecular adaptor activity processes: - preferred_term: double-strand break repair via classical nonhomologous end joining term: id: GO:0097680 label: double-strand break repair via classical nonhomologous end joining locations: - preferred_term: site of double-strand break term: id: GO:0035861 label: site of double-strand break role_description: >- Not on the RGD gene list (identified in 2015, after the RGD diagram); added as the third XRCC4-superfamily NHEJ factor. Its single experimental MF row is the generic molecular adaptor activity. evidence: - source_id: PMID:25574025 statement: PAXX binds Ku directly, promotes Ku-dependent ligation and assembly of core NHEJ factors. supporting_text: "Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells." connections: - source: nhej_upstream_signalling target: ku_end_binding connection_type: POSITIVELY_REGULATES description: 53BP1-RIF1 blocks resection, keeping ends double-stranded and available to Ku. - source: ku_end_binding target: dna_pk_activation connection_type: POSITIVELY_REGULATES description: Ku recruits DNA-PKcs to the ends (RGD draws Xrcc5 and Xrcc6 --+> Prkdc). - source: dna_pk_activation target: end_processing connection_type: POSITIVELY_REGULATES description: DNA-PKcs autophosphorylation releases the ends, and DNA-PKcs phosphorylates and activates Artemis. - source: ku_end_binding target: gap_filling connection_type: POSITIVELY_REGULATES description: Ku recruits pol mu and pol lambda through their BRCT domains. - source: ku_end_binding target: ligation connection_type: POSITIVELY_REGULATES description: Ku recruits XRCC4-LIG4, XLF and PAXX; PAXX binds Ku directly. - source: end_processing target: ligation connection_type: PROVIDES_INPUT_FOR description: Processing produces 5'-phosphate and 3'-hydroxyl termini that LIG4 can seal. - source: gap_filling target: ligation connection_type: PROVIDES_INPUT_FOR description: Filled gaps leave ligatable nicks. knowledge_gaps: - gap_statement: >- Whether APLF's reported endo- and exonuclease activity operates during chromosomal NHEJ, or whether APLF acts only as a poly(ADP-ribose)- and Ku-dependent scaffold that retains XRCC4-LIG4 and chaperones histones, is unresolved. gap_kind: - BIOLOGY - gap_statement: >- How the DNA-PK long-range synaptic complex transitions to the short-range complex in which XRCC4-LIG4, XLF and PAXX align the ends for ligation, and how DNA-PKcs autophosphorylation and the partial redundancy of XLF and PAXX control this transition and end-processing access in cells, is incompletely understood. gap_kind: - BIOLOGY