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

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.

MODULE:eukaryotic_mismatch_repairDRAFTCONCRETEBiological Processmodules/eukaryotic_mismatch_repair.yaml
mismatch repairGO:0006298
PW:0000662
mismatch repair pathway
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).
GO:0006298
mismatch repair
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.
PMID:16143102
Reconstitution of 5'-directed human mismatch repair in a purified system.
Human MMR has been reconstituted from purified components, and the reconstituted protein set defines the module's parts.
The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I.
PMID:16873062
Endonucleolytic function of MutLalpha in human mismatch repair.
MutLalpha is a latent endonuclease activated by the mismatch, MutSalpha, RFC, PCNA and ATP; its incision is biased to the nicked strand.
We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner.
PMID:33510387
Mispair-bound human MutS-MutL complex triggers DNA incisions and activates mismatch repair.
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.
MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch.
PMID:10748105
Identification of a second MutL DNA mismatch repair complex (hPMS1 and hMLH1) in human epithelial cells.
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.
Our data provide evidence that MutL proteins form two different complexes, MutL-alpha (hPMS2 and hMLH1) and MutL-beta (hPMS1 and hMLH1).
PMID:10615123
MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability.
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.
Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability.
PMID:11292842
The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2.
MLH3, PMS1 and PMS2 compete for the same MLH1 interaction region, so MutLbeta and MutLgamma draw on the MLH1 pool that MutLalpha needs.
Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues.

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.

10Nodes
7Parts
1Variant Sets
2Variants
10Annotons
8Connections

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:eukaryotic_mismatch_repair 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

2 gene-function check(s) reflected in core functions · 0 conflict(s) · 4 evidence or coverage gap(s) (13 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
mutsalpha_mismatch_sensor PROTEIN_COMPLEX
Gene 		ATP-dependent DNA damage sensor activity
GO:0140664 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
mutsalpha_mismatch_sensor/mutsalpha_msh6 MSH6
UniProtKB:P52701
Complex Unit 		histone H3K36me3 reader activity
GO:0140003 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
mutsbeta_loop_sensor PROTEIN_COMPLEX
Gene 		mismatched DNA binding
GO:0030983 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
rfc_clamp_loader PROTEIN_COMPLEX
Gene 		DNA clamp loader activity
GO:0003689 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
pcna_clamp PROTEIN_COMPLEX
Gene 		DNA polymerase processivity factor activity
GO:0030337 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
mutlalpha_endonuclease PROTEIN_COMPLEX
Gene 		DNA endonuclease activity
GO:0004520 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
exo1_exonuclease EXO1
UniProtKB:Q9UQ84
Gene 		double-stranded DNA 5'-3' DNA exonuclease activity
GO:0051908 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
hmgb1_excision_cofactor HMGB1
UniProtKB:P09429
Gene 		DNA binding, bending
GO:0008301 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rpa_ssdna_binding PROTEIN_COMPLEX
Gene 		single-stranded DNA binding
GO:0003697 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
pold_polymerase PROTEIN_COMPLEX
Gene 		DNA-directed DNA polymerase activity
GO:0003887 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
pold_polymerase/pold_pold1 POLD1
UniProtKB:P28340
Complex Unit 		DNA-directed DNA polymerase activity
GO:0003887 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
pold_polymerase/pold_pold3 POLD3
UniProtKB:Q15054
Complex Unit 		protein-macromolecule adaptor activity
GO:0030674 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
lig1_ligase LIG1
UniProtKB:P18858
Gene 		DNA ligase activity
GO:0003909 		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.

  • rpa_gap_protection ⊣ exo1_excision [input]
    rpa_gap_protection is a negative regulator with no upstream edge (not described as feedback)

Gene-review completeness (3/21 grounded genes reviewed)

2 complete review(s) · 2 with deep research · 18 missing review · 1 reviewed but lacking deep research

Gene Review Complete Deep research
EXO1 Q9UQ84 ✓ ✓ ✗
HMGB1 P09429 ✓ 213/215 ✓
PCNA P12004 ✗ — —
RPA2 P15927 ✗ — —
LIG1 P18858 ✗ — —
MSH3 P20585 ✗ — —
RPA1 P27694 ✗ — —
POLD1 P28340 ✗ — —
RPA3 P35244 ✗ — —
RFC4 P35249 ✗ — —
RFC2 P35250 ✗ — —
RFC1 P35251 ✗ — —
MLH1 P40692 ✗ — —
RFC5 P40937 ✗ — —
RFC3 P40938 ✗ — —
MSH2 P43246 ✗ — —
MSH6 P52701 ✗ — —
PMS2 P54278 ✗ — —
POLD2 P49005 ✓ ✓ ✓
POLD3 Q15054 ✗ — —
POLD4 Q9HCU8 ✗ — —

Details

Context
Homo sapiensNCBITaxon:9606
nucleusGO:0005634
Human DNA mismatch repairBiological Processeukaryotic_mismatch_repair
mismatch repairGO:0006298
Context
Homo sapiensNCBITaxon:9606
nucleusGO:0005634

Connections

mismatch_recognition -> mutlalpha_incision Positively Regulates
Mismatch-bound, ATP-loaded MutS recruits MutLalpha and is required to activate its endonuclease.
pcna_loading -> mutlalpha_incision Positively Regulates
Oriented PCNA activates MutLalpha and directs incision to the discontinuous strand.
mutlalpha_incision -> exo1_excision Provides Input For
The MutLalpha nick 5' to the mismatch is the entry point for EXO1.
mismatch_recognition -> exo1_excision Positively Regulates
MutSalpha activates EXO1 and confers processivity on it.
rpa_gap_protection -> exo1_excision Negatively Regulates
RPA bound to the gap displaces EXO1 and helps terminate excision past the mismatch.
exo1_excision -> pold_resynthesis Provides Input For
The RPA-coated single-stranded gap is the template for resynthesis.
pcna_loading -> pold_resynthesis Positively Regulates
PCNA is the processivity clamp for POLD.
pold_resynthesis -> lig1_ligation Provides Input For
The nick left after gap filling is sealed by LIG1.
Part 1: mismatch recognition
Mismatch recognition by a MutS homologue heterodimerMolecular Functionmismatch_recognition

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 set: MutS homologue heterodimer by mismatch type (base-base mismatches and small loops versus larger insertion/deletion loops) [Realizer] (One Or More)
PMID:8942985
MSH2-MSH3 and MSH2-MSH6 bind mismatched DNA with overlapping but not identical specificity.
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.
PMID:16143102
In the reconstituted reaction MutSbeta is the preferred sensor for insertion/deletion mispairs.
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.
MutSalpha (MSH2-MSH6)Protein Complexmutsalpha_variant

Annotons

MutSalpha ATP-dependent mismatch sensor
mutsalpha_mismatch_sensor
Participant: Protein Complex: MutSalpha (MSH2-MSH6)
Protein Complex:
MutSalpha (MSH2-MSH6)GO:0032301
Active units:
MSH2 subunit
Participant: Gene Product: MSH2
Gene Product:
MSH2UniProtKB:P43246
Role: Shared MutS-homologue subunit; ATPase and mismatch-binding partner in both MutSalpha and MutSbeta.
MSH6 subunit
Participant: Gene Product: MSH6
Gene Product:
MSH6UniProtKB:P52701
Role: Mismatch-contacting subunit that confers base-base specificity; its PWWP domain reads H3K36me3 and its N-terminal motif binds PCNA.
Function:
histone H3K36me3 reader activityGO:0140003

Function

ATP-dependent DNA damage sensor activityGO:0140664

Processes

mismatch repairGO:0006298

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.

PMID:33510387
Sliding of MutSalpha is needed for final mismatch removal rather than for initiation.
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.
MutSbeta (MSH2-MSH3)Protein Complexmutsbeta_variant

Annotons

MutSbeta insertion/deletion loop recognition
mutsbeta_loop_sensor
Participant: Protein Complex: MutSbeta (MSH2-MSH3)
Protein Complex:
MutSbeta (MSH2-MSH3)GO:0032302
Active units:
MSH2 subunit
Participant: Gene Product: MSH2
Gene Product:
MSH2UniProtKB:P43246
Role: Shared MutS-homologue subunit.
MSH3 subunit
Participant: Gene Product: MSH3
Gene Product:
MSH3UniProtKB:P20585
Role: Loop-specificity subunit; carries an N-terminal PCNA-binding motif.

Function

mismatched DNA bindingGO:0030983

Processes

mismatch repairGO:0006298

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.

Part 2: PCNA loading by RFC (strand discrimination signal)
RFC loading of the PCNA clamp at the strand discontinuityBiological Processpcna_loading

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

RFC (RFC1-RFC5) DNA clamp loader
rfc_clamp_loader
Participant: Protein Complex: replication factor C (RFC1-RFC5)
Protein Complex:
replication factor C (RFC1-RFC5)GO:0005663
Active units:
RFC1 large subunit
Participant: Gene Product: RFC1
Gene Product:
RFC1UniProtKB:P35251
Role: Large subunit; DNA-binding and clamp-interacting.
RFC2 subunit
Participant: Gene Product: RFC2
Gene Product:
RFC2UniProtKB:P35250
Role: AAA+ ATPase small subunit.
RFC3 subunit
Participant: Gene Product: RFC3
Gene Product:
RFC3UniProtKB:P40938
Role: AAA+ ATPase small subunit.
RFC4 subunit
Participant: Gene Product: RFC4
Gene Product:
RFC4UniProtKB:P35249
Role: AAA+ ATPase small subunit.
RFC5 subunit
Participant: Gene Product: RFC5
Gene Product:
RFC5UniProtKB:P40937
Role: AAA+ ATPase small subunit.

Function

DNA clamp loader activityGO:0003689
Targets: PCNAUniProtKB:P12004

Processes

mismatch repairGO:0006298

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.

PMID:16873062
RFC is one of the four factors needed to activate MutLalpha incision.
We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner.
PCNA sliding clamp
pcna_clamp
Participant: Protein Complex: PCNA homotrimer
Protein Complex:
PCNA homotrimerGO:0043626
Active units:
PCNA protomer3
Participant: Gene Product: PCNA
Gene Product:
PCNAUniProtKB:P12004
Role: Forms the homotrimeric ring; binds PIP-box motifs in MSH6, MSH3, MLH1, PMS2 and POLD.

Function

DNA polymerase processivity factor activityGO:0030337

Processes

mismatch repairGO:0006298

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.

PMID:11005803
PCNA binds MSH2-MSH6 and MSH2-MSH3, and the interaction is needed before resynthesis.
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.
Part 3: strand-directed incision
MutLalpha (MLH1-PMS2) endonuclease nicking of the daughter strandReactionmutlalpha_incision

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

MutLalpha latent DNA endonuclease
mutlalpha_endonuclease
Participant: Protein Complex: MutLalpha (MLH1-PMS2)
Protein Complex:
MutLalpha (MLH1-PMS2)GO:0032389
Active units:
MLH1 subunit
Participant: Gene Product: MLH1
Gene Product:
MLH1UniProtKB:P40692
Role: Shared MutL-homologue subunit; ATPase and dimerisation partner of PMS2, PMS1 and MLH3.
PMS2 catalytic subunit
Participant: Gene Product: PMS2
Gene Product:
PMS2UniProtKB:P54278
Role: Carries the endonuclease active-site motif.

Function

DNA endonuclease activityGO:0004520

Processes

mismatch repairGO:0006298

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.

PMID:16873062
The endonuclease active site is in PMS2.
The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif.
PMID:16873062
Incision is directed to the nicked strand.
Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand.
Part 4: excision of the error-containing tract
EXO1 5'-to-3' excision from the nick, stimulated by HMGB1Reactionexo1_excision

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

EXO1 double-stranded DNA 5'-3' exonuclease
exo1_exonuclease
Participant: Gene Product: EXO1
Gene Product:
EXO1UniProtKB:Q9UQ84

Function

double-stranded DNA 5'-3' DNA exonuclease activityGO:0051908

Processes

mismatch repairGO:0006298

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.

PMID:14636568
MutSalpha activates EXO1 and excision stops after the mispair is removed.
In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair.
PMID:11809771
EXO1 is the excision activity of human nuclear extracts.
In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner.
HMGB1 stimulation of MutSalpha-activated excision
hmgb1_excision_cofactor
Participant: Gene Product: HMGB1
Gene Product:
HMGB1UniProtKB:P09429

Function

DNA binding, bendingGO:0008301

Processes

positive regulation of mismatch repairGO:0032425

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.

PMID:15014079
HMGB1 binds MutSalpha and acts before excision.
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.
PMID:16143102
RPA and HMGB1 stimulate excision with complementary roles.
RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch
file:human/HMGB1/HMGB1-ai-review.yaml
The HMGB1 review accepts positive regulation of mismatch repair (IDA, PMID:15014079).
Part 5: single-stranded gap protection
RPA coating of the excision gapMolecular Functionrpa_gap_protection

Annotons

RPA heterotrimer single-stranded DNA binding
rpa_ssdna_binding
Participant: Protein Complex: replication protein A (RPA1-RPA3)
Protein Complex:
replication protein A (RPA1-RPA3)GO:0005662
Active units:
RPA1 (RPA70) subunit
Participant: Gene Product: RPA1
Gene Product:
RPA1UniProtKB:P27694
Role: Main ssDNA-binding subunit; its zinc-finger motif is required for MMR in vitro.
RPA2 (RPA32) subunit
Participant: Gene Product: RPA2
Gene Product:
RPA2UniProtKB:P15927
Role: ssDNA-binding and regulatory (phosphorylated) subunit.
RPA3 (RPA14) subunit
Participant: Gene Product: RPA3
Gene Product:
RPA3UniProtKB:P35244
Role: Structural subunit of the trimerisation core.

Function

single-stranded DNA bindingGO:0003697

Processes

mismatch repairGO:0006298

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).

PMID:9430682
Wild-type RPA is required for mismatch repair in vitro.
We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro.
PMID:16143102
RPA has a distinct role in excision termination.
RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch.
Part 6: gap-filling DNA synthesis
DNA polymerase delta resynthesis with PCNAReactionpold_resynthesis

Annotons

DNA polymerase delta (POLD1-POLD4) gap filling
pold_polymerase
Participant: Protein Complex: DNA polymerase delta holoenzyme (POLD1-POLD4)
Protein Complex:
DNA polymerase delta holoenzyme (POLD1-POLD4)GO:0043625
Active units:
POLD1 catalytic subunit
Participant: Gene Product: POLD1
Gene Product:
POLD1UniProtKB:P28340
Role: Polymerase and 3'-5' proofreading exonuclease.
Function:
DNA-directed DNA polymerase activityGO:0003887
POLD2 (p50) subunit
Participant: Gene Product: POLD2
Gene Product:
POLD2UniProtKB:P49005
Role: Non-catalytic B-subunit bridging POLD1 and POLD3; contributes to the holoenzyme's DNA polymerase activity (POLD2 gene review core function).
POLD3 (p66) subunit
Participant: Gene Product: POLD3
Gene Product:
POLD3UniProtKB:Q15054
Role: Carries a PIP box that tethers the holoenzyme to PCNA.
Function:
protein-macromolecule adaptor activityGO:0030674
POLD4 (p12) subunit
Participant: Gene Product: POLD4
Gene Product:
POLD4UniProtKB:Q9HCU8
Role: Small subunit; degraded after DNA damage. No experimental molecular function in human GOA.

Function

DNA-directed DNA polymerase activityGO:0003887

Processes

mismatch repairGO:0006298

PCNA-dependent POLD fills the excision gap. POLD1 carries this activity by IMP (PMID:16762037). Polymerase epsilon is not part of the reconstituted reaction.

PMID:33510387
POLD fills the gap with PCNA and RPA, and LIG1 seals the nick.
The DNA gap is filled by DNA polymerase δ in concerted reactions with PCNA and RPA, and the nick is ligated by ligase I.
file:human/POLD2/POLD2-ai-review.yaml
The POLD2 gene review covers the accessory subunit's role in the polymerase delta holoenzyme.
Part 7: nick ligation
DNA ligase 1 sealing of the repaired strandReactionlig1_ligation

Annotons

LIG1 DNA ligase
lig1_ligase
Participant: Gene Product: LIG1
Gene Product:
LIG1UniProtKB:P18858

Function

DNA ligase activityGO:0003909

Processes

mismatch repairGO:0006298

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).

PMID:16143102
Ligase I is the ligase of the reconstituted human reaction.
The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I.