id: MODULE:eukaryotic_mismatch_repair title: Human DNA mismatch repair — MutSalpha/MutSbeta mismatch recognition, RFC-PCNA-directed MutLalpha incision, EXO1 excision with RPA protection, and POLD resynthesis with LIG1 ligation; MSH2/MSH6/MSH3/MLH1/PMS2/RFC1-5/PCNA/EXO1/HMGB1/RPA1-3/POLD1-4/LIG1 description: >- Post-replicative DNA mismatch repair (MMR) removes base-base mismatches and small insertion/deletion loops that escape the proofreading of replicative DNA polymerases. In human cells a mismatch is recognised by one of two MutS homologue heterodimers that share MSH2: MutSalpha (MSH2-MSH6) binds base-base mismatches and one- or two-nucleotide insertion/deletion loops, and MutSbeta (MSH2-MSH3) binds larger insertion/deletion loops and has only a limited role in base-base mismatch repair. MutSalpha is pre-enriched on chromatin through binding of the MSH6 PWWP domain to H3K36me3. In an ATP-dependent step the mismatch-bound MutS complex recruits MutLalpha (MLH1-PMS2). MutLalpha is a latent endonuclease whose active site lies in PMS2; it is activated by the mismatch, MutSalpha, ATP and the PCNA sliding clamp that replication factor C (RFC1-5) loads at the strand break or primer terminus. Because RFC loads PCNA in a fixed orientation, the activated MutLalpha nicks the discontinuous (newly synthesised) strand, which is how the daughter strand is identified. Exonuclease 1 (EXO1), activated by MutSalpha, removes the error-containing tract 5' to 3' from a nick, and excision stops after the mispair is removed through the combined action of MutLalpha and RPA, the heterotrimeric single-stranded DNA-binding protein (RPA1-RPA3) that coats and protects the gap. The chromatin protein HMGB1 stimulates MutSalpha-activated excision. The gap is filled by DNA polymerase delta (POLD1-POLD4) working with PCNA and RFC, and DNA ligase 1 (LIG1) seals the remaining nick. Loss of MSH2, MSH6, MLH1 or PMS2 causes microsatellite instability and Lynch syndrome. Mismatch avoidance by polymerase proofreading, MMR-dependent signalling of DNA damage, meiotic crossover resolution and the specialised mutagenic MMR of antibody genes are outside this module. status: DRAFT scope: CONCRETE evidence: - source_id: PW:0000662 title: mismatch repair pathway statement: >- The module boundary follows the RGD Pathway Ontology term and its diagram. The PW definition names Msh proteins (MutS counterparts), Mlh and Pms proteins (MutL counterparts), Exo1 excision, Rpa protection of single-stranded DNA, and resynthesis and ligation by DNA polymerase delta and DNA ligase 1 with the Pcna clamp and the Rfc clamp loader. The diagram draws Exo1, Lig1, the Mlh and Msh members, the Rfc complex and the pold complex associated with Pcna. Derived through projects/RGD_PATHWAY_ONTOLOGY (pathways/PW_0000662.yaml). - source_id: GO:0006298 title: mismatch repair statement: >- skos:closeMatch for PW:0000662 in the project seed (RGD page GO xref, PW definition xref and lexical match). It is the module's process concept. - source_id: PMID:16143102 title: Reconstitution of 5'-directed human mismatch repair in a purified system. statement: >- Human MMR has been reconstituted from purified components, and the reconstituted protein set defines the module's parts. supporting_text: The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. - source_id: PMID:16873062 title: Endonucleolytic function of MutLalpha in human mismatch repair. statement: >- MutLalpha is a latent endonuclease activated by the mismatch, MutSalpha, RFC, PCNA and ATP; its incision is biased to the nicked strand. supporting_text: We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. - source_id: PMID:33510387 title: Mispair-bound human MutS-MutL complex triggers DNA incisions and activates mismatch repair. statement: >- Current model of the human reaction: MutSalpha recruits MutLalpha, which nicks the daughter strand 5' to the mismatch; EXO1 excises from that nick; POLD fills the gap with PCNA and RPA, and LIG1 seals it. supporting_text: MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch. - source_id: PMID:10748105 title: Identification of a second MutL DNA mismatch repair complex (hPMS1 and hMLH1) in human epithelial cells. statement: >- MutLbeta (MLH1-PMS1) exists in human cells, but this study shows only complex formation and MLH1-dependent stability, not a repair activity; MutLbeta is therefore recorded as context, not as a part. supporting_text: Our data provide evidence that MutL proteins form two different complexes, MutL-alpha (hPMS2 and hMLH1) and MutL-beta (hPMS1 and hMLH1). - source_id: PMID:10615123 title: 'MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability.' statement: >- MLH3 binds MLH1 (MutLgamma) and a dominant-negative MLH3 causes microsatellite instability, supporting at most a minor or redundant somatic MMR role; MutLgamma is recorded as context, not as a part. supporting_text: Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. - source_id: PMID:11292842 title: 'The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2.' statement: >- MLH3, PMS1 and PMS2 compete for the same MLH1 interaction region, so MutLbeta and MutLgamma draw on the MLH1 pool that MutLalpha needs. supporting_text: Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. notes: >- Derived from the RGD Pathway Ontology term PW:0000662 (mismatch repair pathway) through the RGD_PATHWAY_ONTOLOGY project (seed in projects/RGD_PATHWAY_ONTOLOGY/data/module_seeds.yaml, tier related_module_only; mapped GO process mismatch repair, skos:closeMatch; the PW page also cross-references KEGG map03430). RGD curates 24 human genes to PW:0000662: EXO1 HMGB1 LIG1 MLH1 MLH3 MSH2 MSH3 MSH6 PCNA PMS1 PMS2 POLD1 POLD2 POLD3 POLD4 RFC1 RFC2 RFC3 RFC4 RFC5 RPA1 RPA2 RPA3 RPA4. Twenty-one are modelled as participants; MLH3, PMS1 and RPA4 are context (decisions 1 to 3). The parts follow the human reaction reconstituted from purified proteins (PMID:16143102, PMID:16873062), which uses exactly the RGD set minus those three. Molecular functions were taken from each protein's experimental GO annotations in QuickGO (November 2026); departures are stated on the annotons (PCNA processivity factor activity is IBA-supported; MutSbeta uses the parent term mismatched DNA binding). Gene reviews exist only for HMGB1 and POLD2. Boundary decisions. (1) MutLbeta (MLH1-PMS1) is context, not a part. It forms in human cells (PMID:10748105), but no repair activity has been shown for it, PMS1 is not needed in the reconstituted reaction, and PMS1 germline variants are not established Lynch syndrome alleles. (2) MutLgamma (MLH1-MLH3) is context, not a part. MLH3 carries the conserved endonuclease motif, and a dominant-negative MLH3 causes microsatellite instability (PMID:10615123), but its established role is meiotic crossover formation, and any somatic MMR role is minor or a backup for MutLalpha; it would be a variant of the incision step only if purified human MutLgamma were shown to support repair. MLH3, PMS1 and PMS2 compete for the same MLH1 region (PMID:11292842). (3) RPA4 is excluded. It is an RPA2 paralogue that forms an alternative RPA complex that does not support DNA replication, and there is no evidence that it acts in MMR; the canonical RPA1-RPA3 heterotrimer is the RPA of the reconstituted reaction (PMID:9430682). (4) HMGB1 is included as an accessory annoton in the excision step, with positive regulation of mismatch repair as its process (accepted in the HMGB1 gene review), because it stimulates MutSalpha-activated excision but is not an essential catalytic participant. (5) EXO1-independent excision is context. Excision from a MutLalpha nick without EXO1, by POLD strand displacement or another nuclease, is described in the literature (PMID:33510387 notes unidentified nucleases) but has no defined human participant set, so it is not modelled as an alternative route and is recorded as a knowledge gap. (6) PCNA and RFC are modelled as one strand-discrimination step that feeds both incision and resynthesis; PCNA is not duplicated in the resynthesis step. (7) Out of scope: polymerase proofreading (DNA replication proofreading), MMR-dependent DNA damage signalling and apoptosis in response to alkylating agents, meiotic mismatch repair and crossover resolution, somatic hypermutation and class-switch recombination (which use MutSalpha, EXO1 and polymerase eta mutagenically), and the MCM9 helicase and WRN, which modulate MMR but are not RGD members. MSH4 and MSH5 are meiotic MutS homologues and are not MMR participants. Related modules. No human DNA replication, PCNA, RFC or polymerase delta module exists in modules/ (searched for replication, PCNA, RFC, polymerase, ligase, clamp and repair). Drosophila modules on the shared machinery exist by name only: dmel_dna_polymerase_complexes, dmel_elg1_rfc_like_complex and dmel_9_1_1_checkpoint_clamp. The bacterial analogue is muth_independent_mismatch_repair (MutS, MutL endonuclease directed by the beta clamp, UvrD, SSB and exonucleases), which shares the mapped GO process but not the taxon; the two are not linked as variants because their participants are not orthologous at the helicase and excision steps. GO-CAM. No GO-CAM model in gocams/index.tsv contains a mismatch repair activity for any member. The seed's best GO-CAM lead (gomodel:5fce9b7300002336, SPModule-TREM1-DAP12) overlaps only through HMGB1 as an extracellular receptor ligand and is unrelated; EXO1 and RPA1 appear only in DNA end-resection and homologous recombination models. No gocam_associations are recorded. Observations for upstream review. (a) PMS1 carries contributes_to DNA endonuclease activity (IDA, PMID:33510387) in human GOA, but the full text of that paper assays human MLH1-PMS2 and mentions PMS1 only as the yeast MutLalpha subunit (yeast MLH1-PMS1), so the row may be a yeast-human naming confusion. (b) PCNA carries purine-specific mismatch base pair DNA N-glycosylase activity (IDA, PMID:11005803), but the abstract of that paper reports PCNA binding to MSH2-MSH6 and MSH2-MSH3 and no glycosylase assay; this looks like a mis-selected term. (c) MLH1 and PMS2 carry meiotic mismatch repair (IDA, ComplexPortal) from PMID:16873062, a study of somatic MMR in human cell extracts; only the abstract was checked. (d) LIG1 has mismatch repair only by TAS and POLD subunits have no experimental mismatch repair row, although both are required in the reconstituted reaction (PMID:16143102). PANTHER family ids and PTNs are deliberately not asserted. module: id: eukaryotic_mismatch_repair label: Human DNA mismatch repair module_type: BIOLOGICAL_PROCESS concepts: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair description: >- The GO process mapped (skos:closeMatch) to PW:0000662. Here the human, nick-directed, post-replicative reaction. context: taxa: - preferred_term: Homo sapiens term: id: NCBITaxon:9606 label: Homo sapiens cellular_components: - preferred_term: nucleus term: id: GO:0005634 label: nucleus parts: - order: 1 role: mismatch recognition node: id: mismatch_recognition label: Mismatch recognition by a MutS homologue heterodimer module_type: MOLECULAR_FUNCTION description: >- MSH2 partners with MSH6 (MutSalpha) or MSH3 (MutSbeta). The two complexes have overlapping but distinct specificities: MutSalpha for base-base mismatches and small loops, MutSbeta for larger insertion/deletion loops. Both carry PCNA-binding motifs in their MSH6 or MSH3 subunit. variant_sets: - id: muts_heterodimer label: MutS homologue heterodimer axis: mismatch type (base-base mismatches and small loops versus larger insertion/deletion loops) variant_kind: REALIZER selection: ONE_OR_MORE evidence: - source_id: PMID:8942985 statement: >- MSH2-MSH3 and MSH2-MSH6 bind mismatched DNA with overlapping but not identical specificity. supporting_text: Analysis of the mismatched nucleotide-binding specificity of the hMSH2-hMSH3 and hMSH2-hMSH6 protein complexes showed that they have overlapping but not identical binding specificity. - source_id: PMID:16143102 statement: In the reconstituted reaction MutSbeta is the preferred sensor for insertion/deletion mispairs. supporting_text: In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. variants: - id: mutsalpha_variant label: MutSalpha (MSH2-MSH6) module_type: PROTEIN_COMPLEX annotons: - id: mutsalpha_mismatch_sensor label: MutSalpha ATP-dependent mismatch sensor participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: MutSalpha (MSH2-MSH6) term: id: GO:0032301 label: MutSalpha complex active_units: - id: mutsalpha_msh2 label: MSH2 subunit role: Shared MutS-homologue subunit; ATPase and mismatch-binding partner in both MutSalpha and MutSbeta. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MSH2 term: id: UniProtKB:P43246 label: DNA mismatch repair protein Msh2 - id: mutsalpha_msh6 label: MSH6 subunit role: >- Mismatch-contacting subunit that confers base-base specificity; its PWWP domain reads H3K36me3 and its N-terminal motif binds PCNA. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MSH6 term: id: UniProtKB:P52701 label: DNA mismatch repair protein Msh6 function: preferred_term: histone H3K36me3 reader activity term: id: GO:0140003 label: histone H3K36me3 reader activity evidence: - source_id: PMID:23622243 statement: The MSH6 PWWP domain binds H3K36me3 and recruits MutSalpha to chromatin. supporting_text: Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. function: preferred_term: ATP-dependent DNA damage sensor activity term: id: GO:0140664 label: ATP-dependent DNA damage sensor activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- Binds a base-base mismatch or a one- or two-nucleotide loop and, after ATP binding, recruits MutLalpha to form the initiation complex; ATP-dependent release from the mismatch later gives EXO1 access. MSH2 and MSH6 carry this activity as contributes_to (IDA) in human GOA. evidence: - source_id: PMID:33510387 statement: Sliding of MutSalpha is needed for final mismatch removal rather than for initiation. supporting_text: Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. - id: mutsbeta_variant label: MutSbeta (MSH2-MSH3) module_type: PROTEIN_COMPLEX annotons: - id: mutsbeta_loop_sensor label: MutSbeta insertion/deletion loop recognition participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: MutSbeta (MSH2-MSH3) term: id: GO:0032302 label: MutSbeta complex active_units: - id: mutsbeta_msh2 label: MSH2 subunit role: Shared MutS-homologue subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MSH2 term: id: UniProtKB:P43246 label: DNA mismatch repair protein Msh2 - id: mutsbeta_msh3 label: MSH3 subunit role: Loop-specificity subunit; carries an N-terminal PCNA-binding motif. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MSH3 term: id: UniProtKB:P20585 label: DNA mismatch repair protein Msh3 function: preferred_term: mismatched DNA binding term: id: GO:0030983 label: mismatched DNA binding processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- Binds insertion/deletion loops of one or more extra nucleotides (+G, +CA, +(CA)5 in the cited gel-shift assay) but not a G/T mismatch. MSH3 carries mismatched DNA binding and its children dinucleotide insertion or deletion binding and dinucleotide repeat insertion binding as contributes_to (IDA, PMID:8942985), and a NOT annotation to guanine/thymine mispair binding; the parent term is used because no experimentally annotated term covers all loop sizes. - order: 2 role: PCNA loading by RFC (strand discrimination signal) node: id: pcna_loading label: RFC loading of the PCNA clamp at the strand discontinuity module_type: BIOLOGICAL_PROCESS description: >- RFC binds PCNA with ATP, opens it and closes it around primer-template or nicked DNA in a fixed orientation. The oriented clamp both activates the MutLalpha endonuclease and directs it to the discontinuous strand, and it is reused as the processivity factor for POLD in resynthesis. PCNA also binds MSH6 and MSH3. annotons: - id: rfc_clamp_loader label: RFC (RFC1-RFC5) DNA clamp loader participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: replication factor C (RFC1-RFC5) term: id: GO:0005663 label: DNA replication factor C complex active_units: - id: rfc_rfc1 label: RFC1 large subunit role: Large subunit; DNA-binding and clamp-interacting. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RFC1 term: id: UniProtKB:P35251 label: Replication factor C subunit 1 - id: rfc_rfc2 label: RFC2 subunit role: AAA+ ATPase small subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RFC2 term: id: UniProtKB:P35250 label: Replication factor C subunit 2 - id: rfc_rfc3 label: RFC3 subunit role: AAA+ ATPase small subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RFC3 term: id: UniProtKB:P40938 label: Replication factor C subunit 3 - id: rfc_rfc4 label: RFC4 subunit role: AAA+ ATPase small subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RFC4 term: id: UniProtKB:P35249 label: Replication factor C subunit 4 - id: rfc_rfc5 label: RFC5 subunit role: AAA+ ATPase small subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RFC5 term: id: UniProtKB:P40937 label: Replication factor C subunit 5 function: preferred_term: DNA clamp loader activity term: id: GO:0003689 label: DNA clamp loader activity targets: - preferred_term: PCNA term: id: UniProtKB:P12004 label: DNA sliding clamp PCNA processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- RFC2-RFC5 carry DNA clamp loader activity as contributes_to (IDA, PMID:12930902, an assay of the RFC2-5 core shared with the CTF18-RFC-like complex). RFC is required for MutLalpha activation. evidence: - source_id: PMID:16873062 statement: RFC is one of the four factors needed to activate MutLalpha incision. supporting_text: We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. - id: pcna_clamp label: PCNA sliding clamp participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: PCNA homotrimer term: id: GO:0043626 label: PCNA complex active_units: - id: pcna_protomer label: PCNA protomer role: Forms the homotrimeric ring; binds PIP-box motifs in MSH6, MSH3, MLH1, PMS2 and POLD. stoichiometry: "3" participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PCNA term: id: UniProtKB:P12004 label: DNA sliding clamp PCNA function: preferred_term: DNA polymerase processivity factor activity term: id: GO:0030337 label: DNA polymerase processivity factor activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- The oriented clamp activates and directs MutLalpha incision and is the processivity factor for POLD during resynthesis. Human GOA has no experimental molecular function for PCNA that captures the MMR role (its experimental MF rows are bindings, plus a purine-specific mismatch DNA N-glycosylase row that looks erroneous); the processivity factor activity used here is IBA-supported. GO has no term for activation of the MutLalpha endonuclease by PCNA. evidence: - source_id: PMID:11005803 statement: PCNA binds MSH2-MSH6 and MSH2-MSH3, and the interaction is needed before resynthesis. supporting_text: When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. - order: 3 role: strand-directed incision node: id: mutlalpha_incision label: MutLalpha (MLH1-PMS2) endonuclease nicking of the daughter strand module_type: REACTION description: >- The MutS-MutLalpha initiation complex, with oriented PCNA, makes incisions on the discontinuous strand, chiefly 5' to the mismatch, giving EXO1 an entry point close to the error. The endonuclease active site is the PMS2 DQHA(X)2E(X)4E motif. annotons: - id: mutlalpha_endonuclease label: MutLalpha latent DNA endonuclease participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: MutLalpha (MLH1-PMS2) term: id: GO:0032389 label: MutLalpha complex active_units: - id: mutlalpha_mlh1 label: MLH1 subunit role: Shared MutL-homologue subunit; ATPase and dimerisation partner of PMS2, PMS1 and MLH3. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: MLH1 term: id: UniProtKB:P40692 label: DNA mismatch repair protein Mlh1 - id: mutlalpha_pms2 label: PMS2 catalytic subunit role: Carries the endonuclease active-site motif. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: PMS2 term: id: UniProtKB:P54278 label: Mismatch repair endonuclease PMS2 function: preferred_term: DNA endonuclease activity term: id: GO:0004520 label: DNA endonuclease activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- MLH1 carries DNA endonuclease activity as contributes_to (IDA, PMID:33510387) and PMS2 as contributes_to (IBA). MutLalpha also terminates EXO1 excision once the mismatch is removed. evidence: - source_id: PMID:16873062 statement: The endonuclease active site is in PMS2. supporting_text: The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. - source_id: PMID:16873062 statement: Incision is directed to the nicked strand. supporting_text: Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. - order: 4 role: excision of the error-containing tract node: id: exo1_excision label: EXO1 5'-to-3' excision from the nick, stimulated by HMGB1 module_type: REACTION description: >- EXO1, activated and made processive by MutSalpha, hydrolyses the incised strand 5' to 3' from a nick and past the mismatch. Excision is terminated by MutLalpha and by RPA after the mispair is removed. HMGB1 stimulates MutSalpha-activated excision. EXO1-independent excision, which explains the weak mutator phenotype of EXO1 loss, is recorded in notes as context and not modelled as a route. annotons: - id: exo1_exonuclease label: EXO1 double-stranded DNA 5'-3' exonuclease participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EXO1 term: id: UniProtKB:Q9UQ84 label: Exonuclease 1 function: preferred_term: double-stranded DNA 5'-3' DNA exonuclease activity term: id: GO:0051908 label: double-stranded DNA 5'-3' DNA exonuclease activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- The only exonuclease in the reconstituted human reaction. EXO1 carries this activity by IDA (PMID:10608837, PMID:11842105) and mismatch repair by IDA and IMP. evidence: - source_id: PMID:14636568 statement: MutSalpha activates EXO1 and excision stops after the mispair is removed. supporting_text: In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair. - source_id: PMID:11809771 statement: EXO1 is the excision activity of human nuclear extracts. supporting_text: In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. - id: hmgb1_excision_cofactor label: HMGB1 stimulation of MutSalpha-activated excision participant: selector_type: GENE_PRODUCT gene_product: preferred_term: HMGB1 term: id: UniProtKB:P09429 label: High mobility group protein B1 function: preferred_term: DNA binding, bending term: id: GO:0008301 label: DNA binding, bending processes: - preferred_term: positive regulation of mismatch repair term: id: GO:0032425 label: positive regulation of mismatch repair role_description: >- Accessory chromatin protein that binds MutSalpha and is required before excision in depleted extracts; it acts with RPA to stimulate excision. Its DNA bending activity (IMP, PMID:19223331) was shown in a transcriptional setting, and how it acts in MMR is not known, so the annoton carries the regulatory process accepted in the HMGB1 review rather than a core MMR process. evidence: - source_id: PMID:15014079 statement: HMGB1 binds MutSalpha and acts before excision. supporting_text: Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair. - source_id: PMID:16143102 statement: RPA and HMGB1 stimulate excision with complementary roles. supporting_text: RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch - source_id: file:human/HMGB1/HMGB1-ai-review.yaml statement: The HMGB1 review accepts positive regulation of mismatch repair (IDA, PMID:15014079). - order: 5 role: single-stranded gap protection node: id: rpa_gap_protection label: RPA coating of the excision gap module_type: MOLECULAR_FUNCTION annotons: - id: rpa_ssdna_binding label: RPA heterotrimer single-stranded DNA binding participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: replication protein A (RPA1-RPA3) term: id: GO:0005662 label: DNA replication factor A complex active_units: - id: rpa_rpa1 label: RPA1 (RPA70) subunit role: Main ssDNA-binding subunit; its zinc-finger motif is required for MMR in vitro. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RPA1 term: id: UniProtKB:P27694 label: Replication protein A 70 kDa DNA-binding subunit - id: rpa_rpa2 label: RPA2 (RPA32) subunit role: ssDNA-binding and regulatory (phosphorylated) subunit. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RPA2 term: id: UniProtKB:P15927 label: Replication protein A 32 kDa subunit - id: rpa_rpa3 label: RPA3 (RPA14) subunit role: Structural subunit of the trimerisation core. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RPA3 term: id: UniProtKB:P35244 label: Replication protein A 14 kDa subunit function: preferred_term: single-stranded DNA binding term: id: GO:0003697 label: single-stranded DNA binding processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- Coats the excision gap, stimulates excision, helps terminate it past the mismatch, and supports resynthesis by POLD. RPA1, RPA2 and RPA3 each carry single-stranded DNA binding (IDA) and mismatch repair (IMP, PMID:9430682). evidence: - source_id: PMID:9430682 statement: Wild-type RPA is required for mismatch repair in vitro. supporting_text: We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. - source_id: PMID:16143102 statement: RPA has a distinct role in excision termination. supporting_text: RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. - order: 6 role: gap-filling DNA synthesis node: id: pold_resynthesis label: DNA polymerase delta resynthesis with PCNA module_type: REACTION annotons: - id: pold_polymerase label: DNA polymerase delta (POLD1-POLD4) gap filling participant: selector_type: PROTEIN_COMPLEX protein_complex: preferred_term: DNA polymerase delta holoenzyme (POLD1-POLD4) term: id: GO:0043625 label: delta DNA polymerase complex active_units: - id: pold_pold1 label: POLD1 catalytic subunit role: Polymerase and 3'-5' proofreading exonuclease. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLD1 term: id: UniProtKB:P28340 label: DNA polymerase delta catalytic subunit function: preferred_term: DNA-directed DNA polymerase activity term: id: GO:0003887 label: DNA-directed DNA polymerase activity - id: pold_pold2 label: POLD2 (p50) subunit role: Non-catalytic B-subunit bridging POLD1 and POLD3; contributes to the holoenzyme's DNA polymerase activity (POLD2 gene review core function). participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLD2 term: id: UniProtKB:P49005 label: DNA polymerase delta subunit 2 - id: pold_pold3 label: POLD3 (p66) subunit role: Carries a PIP box that tethers the holoenzyme to PCNA. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLD3 term: id: UniProtKB:Q15054 label: DNA polymerase delta subunit 3 function: preferred_term: protein-macromolecule adaptor activity term: id: GO:0030674 label: protein-macromolecule adaptor activity - id: pold_pold4 label: POLD4 (p12) subunit role: Small subunit; degraded after DNA damage. No experimental molecular function in human GOA. participant: selector_type: GENE_PRODUCT gene_product: preferred_term: POLD4 term: id: UniProtKB:Q9HCU8 label: DNA polymerase delta subunit 4 function: preferred_term: DNA-directed DNA polymerase activity term: id: GO:0003887 label: DNA-directed DNA polymerase activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- PCNA-dependent POLD fills the excision gap. POLD1 carries this activity by IMP (PMID:16762037). Polymerase epsilon is not part of the reconstituted reaction. evidence: - source_id: PMID:33510387 statement: POLD fills the gap with PCNA and RPA, and LIG1 seals the nick. supporting_text: The DNA gap is filled by DNA polymerase δ in concerted reactions with PCNA and RPA, and the nick is ligated by ligase I. - source_id: file:human/POLD2/POLD2-ai-review.yaml statement: The POLD2 gene review covers the accessory subunit's role in the polymerase delta holoenzyme. - order: 7 role: nick ligation node: id: lig1_ligation label: DNA ligase 1 sealing of the repaired strand module_type: REACTION annotons: - id: lig1_ligase label: LIG1 DNA ligase participant: selector_type: GENE_PRODUCT gene_product: preferred_term: LIG1 term: id: UniProtKB:P18858 label: DNA ligase 1 function: preferred_term: DNA ligase activity term: id: GO:0003909 label: DNA ligase activity processes: - preferred_term: mismatch repair term: id: GO:0006298 label: mismatch repair role_description: >- Seals the final nick. LIG1 carries DNA ligase activity by IDA and IMP; the ATP-dependent child term DNA ligase (ATP) activity is IBA-only in human GOA. Its mismatch repair annotation is TAS (Reactome). evidence: - source_id: PMID:16143102 statement: Ligase I is the ligase of the reconstituted human reaction. supporting_text: The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. connections: - source: mismatch_recognition target: mutlalpha_incision connection_type: POSITIVELY_REGULATES description: Mismatch-bound, ATP-loaded MutS recruits MutLalpha and is required to activate its endonuclease. - source: pcna_loading target: mutlalpha_incision connection_type: POSITIVELY_REGULATES description: Oriented PCNA activates MutLalpha and directs incision to the discontinuous strand. - source: mutlalpha_incision target: exo1_excision connection_type: PROVIDES_INPUT_FOR description: The MutLalpha nick 5' to the mismatch is the entry point for EXO1. - source: mismatch_recognition target: exo1_excision connection_type: POSITIVELY_REGULATES description: MutSalpha activates EXO1 and confers processivity on it. - source: rpa_gap_protection target: exo1_excision connection_type: NEGATIVELY_REGULATES description: RPA bound to the gap displaces EXO1 and helps terminate excision past the mismatch. - source: exo1_excision target: pold_resynthesis connection_type: PROVIDES_INPUT_FOR description: The RPA-coated single-stranded gap is the template for resynthesis. - source: pcna_loading target: pold_resynthesis connection_type: POSITIVELY_REGULATES description: PCNA is the processivity clamp for POLD. - source: pold_resynthesis target: lig1_ligation connection_type: PROVIDES_INPUT_FOR description: The nick left after gap filling is sealed by LIG1. knowledge_gaps: - gap_statement: >- How the mismatch-bound MutS-MutLalpha complex communicates with the strand discontinuity that may lie hundreds of nucleotides away (MutS sliding, DNA looping, or MutLalpha recruitment by PCNA at the primer terminus) is not resolved, and nor is the strand signal on the leading strand, where no Okazaki-fragment ends exist (ribonucleotide-removal nicks have been proposed). gap_kind: - BIOLOGY - gap_statement: >- The identity and relative weight of EXO1-independent excision in human cells are unknown: whether it relies on strand displacement by POLD from MutLalpha nicks, on another nuclease, or on both, and why EXO1 loss gives only a mild mutator phenotype whereas MSH2, MLH1 or PMS2 loss gives a strong one. gap_kind: - BIOLOGY - gap_statement: >- Whether MutLgamma (MLH1-MLH3), which carries a conserved endonuclease motif, or MutLbeta (MLH1-PMS1) contributes any catalytic activity to somatic human mismatch repair, beyond competing with PMS2 for MLH1, has not been shown with purified human proteins. gap_kind: - BIOLOGY