Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0061630 ubiquitin protein ligase activity	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetically inferred RING-type ubiquitin ligase activity. This is the core molecular function of RFWD3 and is strongly corroborated by direct biochemistry. Reason: RFWD3 is a RING-finger E3 ligase (EC 2.3.2.27) with catalytic Cys315; multiple IDA studies demonstrate ligase activity toward RPA, RAD51 and p53. The IBA is at the correct level of specificity and represents the central evolved function. Supporting Evidence: PMID:26474068 We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination.
GO:0016567 protein ubiquitination	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetically inferred involvement in protein ubiquitination, the process carried out by the RFWD3 ligase activity. Reason: Directly supported: RFWD3 ubiquitinates RPA, RAD51 and p53 and promotes ubiquitination of proteins on ssDNA. Correct and well-supported. Supporting Evidence: PMID:33321094 the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA
GO:0005634 nucleus	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetically inferred nuclear localization; RFWD3 acts in the nucleus at replication forks and DNA damage sites. Reason: RFWD3 functions in the nucleus where it ubiquitinates RPA/RAD51 at chromatin; is_active_in nucleus is the correct core location. Supporting Evidence: PMID:28691929 impaired relocation of mutant RFWD3 to chromatin
GO:0031297 replication fork processing	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetically inferred role at stalled replication forks, consistent with RFWD3 promoting fork restart and repair at stalled forks. Reason: RFWD3 is recruited to stalled replication forks and is required for fork restart and repair at stalled forks (Elia et al. 2015). Supporting Evidence: PMID:26474068 RFWD3 is necessary for replication fork restart, normal repair kinetics during
GO:0090734 site of DNA damage	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetically inferred activity at sites of DNA damage; RFWD3 is recruited to and functions at DNA damage sites. Reason: RFWD3 translocates to tracks of laser micro-irradiation and to MMC-induced foci that co-localize with RPA and gamma-H2AX; this is a core functional location. Supporting Evidence: PMID:28575657 translocates to tracks of laser micro-irradiation and co-localizes with the phosphorylated form of histone variant H2AX
GO:0004842 ubiquitin-protein transferase activity	IEA GO_REF:0000002	ACCEPT	Summary: InterPro-based electronic annotation of ubiquitin-protein transferase activity, a parent/equivalent of the demonstrated RING ligase activity. Reason: Correct: RFWD3 is a ubiquitin transferase (RING E3). Slightly more general than GO:0061630 but not wrong; the specific ligase activity is captured by the IDA annotations.
GO:0005634 nucleus	IEA GO_REF:0000120	ACCEPT	Summary: Electronic annotation of nuclear localization, consistent with experimental data. Reason: Nuclear localization is experimentally established (multiple EXP/IDA annotations); the IEA is correct.
GO:0005737 cytoplasm	IEA GO_REF:0000044	KEEP AS NON CORE	Summary: Electronic subcellular-location annotation of cytoplasm; RFWD3 is found in the cytoplasm of undamaged cells before nuclear recruitment upon damage. Reason: A cytoplasmic pool is documented in undamaged cells, but the functional site of RFWD3 is the nucleus/chromatin; cytoplasmic localization is peripheral to its DNA-repair function. Supporting Evidence: PMID:21558276 RFWD3 associates with replication protein
GO:0016567 protein ubiquitination	IEA GO_REF:0000120	ACCEPT	Summary: Electronic annotation of protein ubiquitination, matching experimental evidence. Reason: RFWD3 ubiquitinates multiple substrates; the process annotation is correct.
GO:0016605 PML body	IEA GO_REF:0000044	KEEP AS NON CORE	Summary: Electronic subcellular-location annotation of PML nuclear body, from the reported partial association of RFWD3 with PML bodies in undamaged cells. Reason: RFWD3 is partially associated with PML nuclear bodies in undamaged cells, but this is not the site of its characterized DNA-repair ligase activity. Supporting Evidence: PMID:21558276 RFWD3 associates with replication protein
GO:0036297 interstrand cross-link repair	IEA GO_REF:0000002	ACCEPT	Summary: InterPro-based annotation of interstrand cross-link repair, a core RFWD3 process confirmed experimentally. Reason: RFWD3-deficient cells are profoundly hypersensitive to ICL-inducing agents; ICL repair is a major RFWD3 function (FANCW). Supporting Evidence: PMID:28575657 show profound defects in ICL repair
GO:0061630 ubiquitin protein ligase activity	IEA GO_REF:0000003	ACCEPT	Summary: EC-mapping-based annotation of ubiquitin ligase activity (EC 2.3.2.27). Reason: Consistent with the RecName EC 2.3.2.27 and direct biochemical demonstration of RING E3 ligase activity.
GO:0005515 protein binding	IPI PMID:19549727 Analysis of the human E2 ubiquitin conjugating enzyme protei...	MARK AS OVER ANNOTATED	Summary: IntAct-curated interaction with the E2 conjugating enzyme UBE2N (UBC13) from a systematic E2 interaction-network screen. Reason: GO:0005515 protein binding is uninformative as a molecular function. The biologically relevant interaction (RFWD3 partnering with the K63-forming E2 UBE2N) is better captured by the ligase activity core function; no specific MF term is warranted from this high-throughput screen.
GO:0005515 protein binding	IPI PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	MARK AS OVER ANNOTATED	Summary: IntAct-curated interactions with p53 (TP53) and MDM2 from the RFWD3-MDM2-p53 study. Reason: Generic protein binding is uninformative; the specific and informative interactions are separately annotated as p53 binding (GO:0002039) and MDM2/MDM4 family protein binding (GO:0097371).
GO:0005515 protein binding	IPI PMID:24126761 hPrimpol1/CCDC111 is a human DNA primase-polymerase required...	MARK AS OVER ANNOTATED	Summary: IntAct-curated RFWD3-RPA2 (P15927) interaction detected in a PRIMPOL study. Reason: Uninformative generic binding term; the functionally important RPA2 interaction underlies the core RFWD3 recruitment/ubiquitination function and is captured there.
GO:0005515 protein binding	IPI PMID:25260751 The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in respon...	MARK AS OVER ANNOTATED	Summary: IntAct-curated RFWD3-UBE2N (P61088) interaction detected in a MEKK1/TAB1 study. Reason: Generic protein binding is uninformative; the UBE2N (E2) partnership is subsumed by the ligase activity annotation.
GO:0005515 protein binding	IPI PMID:28691929 Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanc...	MARK AS OVER ANNOTATED	Summary: IntAct-curated RFWD3-RPA2 (P15927) interaction from the FANCW paper. Reason: Uninformative generic term; the RPA2 interaction that recruits RFWD3 to chromatin is central and captured by the core function rather than by GO:0005515.
GO:0005515 protein binding	IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func...	MARK AS OVER ANNOTATED	Summary: IntAct-curated RFWD3-RPA2 (P15927) interaction from a systems-level multimodal cell-mapping study. Reason: Generic protein binding is uninformative; the RPA2 interaction is already represented functionally elsewhere.
GO:0005654 nucleoplasm	IDA GO_REF:0000052	ACCEPT	Summary: HPA immunofluorescence localization to the nucleoplasm. Reason: Consistent with the established nuclear localization; nucleoplasm is an accurate sub-nuclear location.
GO:0005829 cytosol	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: HPA immunofluorescence localization to the cytosol. Reason: A cytosolic pool is consistent with cytoplasmic localization in undamaged cells, but is peripheral to the nuclear DNA-repair function.
GO:0005634 nucleus	EXP PMID:21504906 E3 ligase RFWD3 participates in replication checkpoint contr...	ACCEPT	Summary: Experimental nuclear localization; RFWD3 is recruited to the nucleus/stalled forks upon replication stress. Reason: Nuclear localization is well established experimentally. Supporting Evidence: PMID:21504906 RFWD3 is recruited to stalled replication forks and co-localizes with RPA2 in response to replication stress
GO:0005634 nucleus	EXP PMID:21558276 RING finger and WD repeat domain 3 (RFWD3) associates with r...	ACCEPT	Summary: Experimental nuclear localization from the RFWD3-RPA association study. Reason: Consistent with established nuclear localization.
GO:0005634 nucleus	EXP PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: Experimental nuclear localization; GFP-RFWD3 forms nuclear foci/tracks at DNA damage sites. Reason: Nuclear localization at DNA damage sites is directly demonstrated. Supporting Evidence: PMID:28575657 translocates to tracks of laser micro-irradiation
GO:0005737 cytoplasm	EXP PMID:21558276 RING finger and WD repeat domain 3 (RFWD3) associates with r...	KEEP AS NON CORE	Summary: Experimental cytoplasmic localization; RFWD3 is found in the cytoplasm of undamaged cells. Reason: Real localization in undamaged cells but peripheral to the nuclear repair function.
GO:0005737 cytoplasm	EXP PMID:28691929 Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanc...	KEEP AS NON CORE	Summary: Experimental cytoplasmic localization reported in the FANCW study. Reason: Cytoplasmic pool documented; the functionally critical relocation is to chromatin/nucleus.
GO:0036297 interstrand cross-link repair	IDA PMID:33321094 The ubiquitin ligase RFWD3 is required for translesion DNA s...	ACCEPT	Summary: Direct demonstration of RFWD3's role in the crosslink/lesion-bypass response; RFWD3 promotes ssDNA-protein ubiquitination required for TLS during ICL repair. Reason: RFWD3 is required for translesion synthesis, an integral gap-filling step of ICL repair; ICL repair involvement is a core function. Supporting Evidence: PMID:33321094 TLS across different DNA lesions is drastically impaired
GO:0061630 ubiquitin protein ligase activity	IDA PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	ACCEPT	Summary: Direct demonstration of RFWD3 E3 ligase activity toward p53 (in the RFWD3-MDM2 complex). Reason: In vitro experiments show RFWD3 acts as a p53 E3 ligase; supports the core RING ligase activity. (The p53 process context is secondary.) Supporting Evidence: PMID:20173098 RFWD3 is a p53 E3 ubiquitin ligase
GO:0061630 ubiquitin protein ligase activity	IDA PMID:26474068 RFWD3-Dependent Ubiquitination of RPA Regulates Repair at St...	ACCEPT	Summary: Direct demonstration that RFWD3 ligase activity ubiquitinates RPA. Reason: Core catalytic function; RFWD3 mediates RPA ubiquitination. Supporting Evidence: PMID:26474068 the E3 ligase RFWD3 is responsible for RPA ubiquitination
GO:0061630 ubiquitin protein ligase activity	IDA PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: Direct demonstration of RFWD3 ligase activity; the recombinant C315A active-site mutant is inactive in vitro. Reason: Confirms catalytic RING E3 activity dependent on Cys315; core molecular function. Supporting Evidence: PMID:28575657 which renders the recombinant protein inactive in vitro
GO:0061630 ubiquitin protein ligase activity	IDA PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: Direct demonstration that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Reason: Core catalytic function toward the physiological substrates RPA and RAD51. Supporting Evidence: PMID:28575658 RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo
GO:0000724 double-strand break repair via homologous recombination	IMP PMID:28691929 Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanc...	ACCEPT	Summary: Loss-of-function (patient/engineered mutations) disrupts homologous recombination, establishing RFWD3's role in HR. Reason: HR was disrupted in RFWD3-mutant cells; a core process for RFWD3 (FANCW). Supporting Evidence: PMID:28691929 HR was disrupted in RFWD3-mutant cells
GO:0005634 nucleus	IMP PMID:28691929 Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanc...	ACCEPT	Summary: Mutation-based evidence that RFWD3 relocation to the nucleus/chromatin is required for function. Reason: Nuclear/chromatin relocation is impaired by FA mutations, underscoring the nucleus as the functional compartment. Supporting Evidence: PMID:28691929 impaired relocation of mutant RFWD3 to chromatin
GO:0000724 double-strand break repair via homologous recombination	IDA PMID:26474068 RFWD3-Dependent Ubiquitination of RPA Regulates Repair at St...	ACCEPT	Summary: RFWD3 is required for homologous recombination at stalled replication forks. Reason: Core process; RFWD3 stimulates HR at stalled forks. Supporting Evidence: PMID:26474068 homologous recombination (HR) at stalled replication forks
GO:0000724 double-strand break repair via homologous recombination	IDA PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: RFWD3-deficient cells show HR defects (PARP-inhibitor hypersensitivity, persistent RPA in RAD51 foci). Reason: Supports the core HR function; RFWD3 controls RPA/RAD51 dynamics required for HR. Supporting Evidence: PMID:28575657 unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells
GO:0000724 double-strand break repair via homologous recombination	IDA PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: RFWD3-mediated removal of RPA and RAD51 facilitates late-phase homologous recombination. Reason: Core HR process, mechanistically defined via RPA/RAD51 turnover. Supporting Evidence: PMID:28575658 crucial for progression to the late-phase HR and suppression of the FA phenotype
GO:0005515 protein binding	IPI PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	MARK AS OVER ANNOTATED	Summary: IntAct-curated interactions with RPA2 (P15927) and RAD51 (Q06609), the physiological RFWD3 substrates/partners. Reason: Generic protein binding is uninformative as an MF; the functionally meaningful RPA2 and RAD51 interactions underpin the core ligase/HR function and are captured there.
GO:0005515 protein binding	IPI PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	MARK AS OVER ANNOTATED	Summary: IntAct-curated RFWD3-RPA2 (P15927) interaction. Reason: Uninformative generic binding term; the RPA2 interaction is functionally represented by the core function.
GO:0006974 DNA damage response	IDA PMID:26474068 RFWD3-Dependent Ubiquitination of RPA Regulates Repair at St...	ACCEPT	Summary: RFWD3 acts within the DNA damage response (RPA ubiquitination upon replication stress). Reason: Accurate high-level process; RFWD3's ligase activity is part of the DDR. More specific processes (HR, ICL repair, fork processing) are also annotated. Supporting Evidence: PMID:26474068 to profile ubiquitination in the DNA damage response (DDR)
GO:0006974 DNA damage response	IDA PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: RFWD3 recruitment and action at DNA damage sites within the DDR. Reason: Correct high-level process annotation, consistent with damage-site recruitment. Supporting Evidence: PMID:28575657 translocates to tracks of laser micro-irradiation
GO:0006974 DNA damage response	IDA PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: RFWD3 acts in the DDR via ATR/ATM-regulated ubiquitination of RPA and RAD51. Reason: Correct high-level DDR process; RFWD3 activity depends on ATR/ATM phosphorylation. Supporting Evidence: PMID:28575658 Phosphorylation by ATR and ATM kinases is required for this activity in vivo
GO:0016567 protein ubiquitination	IDA PMID:26474068 RFWD3-Dependent Ubiquitination of RPA Regulates Repair at St...	ACCEPT	Summary: Direct evidence of RFWD3-mediated RPA ubiquitination. Reason: Core process; RFWD3 ubiquitinates RPA on chromatin. Supporting Evidence: PMID:26474068 the E3 ligase RFWD3 is responsible for RPA ubiquitination
GO:0016567 protein ubiquitination	IDA PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: RFWD3 ubiquitinates RPA at sites of ICL-induced fork stalling. Reason: Core process; RFWD3 ubiquitylates RPA. Supporting Evidence: PMID:28575657 interacts with and ubiquitylates replication protein A (RPA)
GO:0016567 protein ubiquitination	IDA PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: RFWD3 polyubiquitinates RPA and RAD51. Reason: Core process, defined toward physiological substrates. Supporting Evidence: PMID:28575658 RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo
GO:0031297 replication fork processing	IDA PMID:26474068 RFWD3-Dependent Ubiquitination of RPA Regulates Repair at St...	ACCEPT	Summary: RFWD3 promotes restart and remodeling of stalled replication forks and supports normal fork progression. Reason: Direct evidence that RFWD3 is necessary for fork restart and repair at stalled forks (Elia et al. 2015); further corroborated by its role in normal fork progression via PCNA binding (Lin et al. 2018) and in ZRANB3-dependent fork remodeling/reversal (Moore et al. 2023). Supporting Evidence: PMID:26474068 we found that depletion of RFWD3 decreased fork restart PMID:30530694 in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif PMID:37036693 RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner
GO:0036297 interstrand cross-link repair	IMP PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: RFWD3-hypomorphic cells are profoundly hypersensitive to ICL-inducing agents, demonstrating an ICL-repair role. Reason: Loss-of-function evidence establishes ICL repair as a major RFWD3 function (core). Supporting Evidence: PMID:28575657 argues strongly that ICL repair is a major function of RFWD3
GO:0036297 interstrand cross-link repair	IMP PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: RFWD3 inactivation impairs ICL repair; suppression of the FA phenotype depends on RFWD3-mediated RPA/RAD51 removal. Reason: Core ICL-repair process consistent with FANCW identity. Supporting Evidence: PMID:28575658 suppression of the FA phenotype
GO:0090734 site of DNA damage	IDA PMID:28575657 RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for...	ACCEPT	Summary: RFWD3 localizes to sites of ICL/DNA damage (laser tracks, MMC foci). Reason: Core functional location, directly imaged. Supporting Evidence: PMID:28575657 translocates to tracks of laser micro-irradiation
GO:0090734 site of DNA damage	IDA PMID:28575658 RFWD3-Mediated Ubiquitination Promotes Timely Removal of Bot...	ACCEPT	Summary: RFWD3 acts at DNA damage sites to remove RPA and RAD51. Reason: Consistent with damage-site localization and function; core functional location. Supporting Evidence: PMID:28575658 timely removal of RPA and RAD51 from DNA damage sites
GO:0005515 protein binding	IPI PMID:21504906 E3 ligase RFWD3 participates in replication checkpoint contr...	MARK AS OVER ANNOTATED	Summary: IntAct-curated direct RFWD3-RPA2 (P15927) interaction, shown with purified proteins. Reason: Generic protein binding is uninformative; the direct RPA2 interaction that recruits RFWD3 to stalled forks is central and represented by the core function.
GO:0035861 site of double-strand break	IDA PMID:21504906 E3 ligase RFWD3 participates in replication checkpoint contr...	ACCEPT	Summary: RFWD3 co-localizes with RPA2 at stalled forks/damage sites in response to replication stress. Reason: Accurate colocalization annotation consistent with recruitment to damage sites; complements the site of DNA damage annotations. Supporting Evidence: PMID:21504906 RFWD3 is recruited to stalled replication forks and co-localizes with RPA2 in response to replication stress
GO:2000001 regulation of DNA damage checkpoint	IMP PMID:21504906 E3 ligase RFWD3 participates in replication checkpoint contr...	KEEP AS NON CORE	Summary: RFWD3 depletion decreases ATR-dependent CHK1 activation after replication stress, implicating it in replication-checkpoint control. Reason: A genuine but secondary/context-dependent role; the effect on CHK1 activation is cell-type dependent and is distinct from the core RPA/RAD51-ubiquitination repair function. Supporting Evidence: PMID:21504906 RFWD3 is important for ATR-dependent Chk1 activation in response to replication stress
GO:0002039 p53 binding	IPI PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	KEEP AS NON CORE	Summary: Direct interaction with p53 (TP53) within the RFWD3-MDM2-p53 complex that regulates p53 stability. Reason: An informative, specific binding (unlike generic protein binding), but it underlies the secondary p53-stabilization role rather than the core DNA-repair ligase function. Supporting Evidence: PMID:20173098 forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53
GO:0005634 nucleus	IDA PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	ACCEPT	Summary: Experimental nuclear localization in the p53-regulation study. Reason: Consistent with established nuclear localization.
GO:0010212 response to ionizing radiation	IDA PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	KEEP AS NON CORE	Summary: RFWD3 is phosphorylated and stabilizes p53 in the late response to ionizing radiation. Reason: A real damage-response context, but tied to the secondary p53 role; the core RFWD3 function is better described by HR/ICL-repair processes. Supporting Evidence: PMID:20173098 p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination
GO:0016567 protein ubiquitination	IDA PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	ACCEPT	Summary: RFWD3 ubiquitinates p53 (with MDM2) in the late DNA-damage response. Reason: A genuine protein-ubiquitination event catalyzed by RFWD3; correct process term, here in the secondary p53 context. Supporting Evidence: PMID:20173098 forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53
GO:0031571 mitotic G1 DNA damage checkpoint signaling	IMP PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	KEEP AS NON CORE	Summary: RFWD3 positively regulates p53 stability when the G1 cell-cycle checkpoint is activated. Reason: Secondary function via p53/MDM2; distinct from the core replication-associated repair activity and supported by a single group. Supporting Evidence: PMID:20173098 a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated
GO:0097371 MDM2/MDM4 family protein binding	IPI PMID:20173098 RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53...	KEEP AS NON CORE	Summary: Direct interaction with MDM2 in the p53-regulatory RFWD3-MDM2 complex. Reason: Informative specific binding supporting the secondary p53-stabilization role, not the core DNA-repair ligase function. Supporting Evidence: PMID:20173098 RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53

Core Functions

RING-type E3 ubiquitin-protein ligase that is recruited via its C-terminal WD40 domain to RPA-coated single-stranded DNA at stalled replication forks and sites of DNA damage, where (following ATM/ATR phosphorylation) it polyubiquitinates the RPA complex and RAD51 to drive their VCP/p97-dependent turnover and timely removal, enabling homologous recombination and interstrand crosslink repair to progress.

Molecular Function:

ubiquitin protein ligase activity

Directly Involved In:

interstrand cross-link repair double-strand break repair via homologous recombination protein ubiquitination

Cellular Locations:

site of DNA damage nucleus

Supporting Evidence:

PMID:26474068
the E3 ligase RFWD3 is responsible for RPA ubiquitination
PMID:28575658
facilitates timely removal
PMID:28575657
interacts with and ubiquitylates replication protein A (RPA)

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000003

Gene Ontology annotation based on Enzyme Commission mapping

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

GO_REF:0000052

Gene Ontology annotation based on curation of immunofluorescence data

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:19549727

Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network.

PMID:20173098

RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage.

PMID:21504906

E3 ligase RFWD3 participates in replication checkpoint control.

PMID:21558276

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

PMID:24126761

hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.

PMID:25260751

The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines.

PMID:26474068

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PMID:28575657

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

PMID:28575658

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

PMID:28691929

Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia.

PMID:30530694

PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication.

PMID:33321094

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

PMID:37036693

RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

PMID:40205054

Multimodal cell maps as a foundation for structural and functional genomics.

Deep Research

Affinage

(RFWD3-deep-research-affinage.md)

Affinage mechanistic annotation for RFWD3 (human) Affinage Affinage (Claude Sonnet reading pass + Opus synthesis pass) 17 citations

Affinage mechanistic annotation for RFWD3 (human)

Current model (mechanistic narrative)

RFWD3 is a RING-finger/WD40 E3 ubiquitin ligase that governs the resolution and restart of stalled and damaged DNA replication forks by remodeling the protein composition of single-stranded DNA at sites of replication stress [PMID:26474068, PMID:33321094]. It is recruited to damaged chromatin and stalled forks through a direct interaction between its C-terminal coiled-coil/WD40 region and the RPA2 subunit of RPA, an interaction required for ATR-dependent Chk1 activation and for localization to interstrand crosslink (ICL)-stalled forks [PMID:21558276, PMID:21504906, PMID:28575657]. At ongoing forks RFWD3 also associates with PCNA via a PIP motif, an interaction that stabilizes the ligase and supports normal fork progression PMID:30530694. RFWD3 catalyzes multi-site polyubiquitination of the RPA complex and of RAD51, modifications that depend on ATR/ATM phosphorylation of RFWD3 and drive VCP/p97-mediated extraction of these factors from DNA, enabling late-phase homologous recombination, fork restart, and ICL repair [PMID:26474068, PMID:28575658]. By promoting PCNA ubiquitination, RFWD3 licenses translesion synthesis and recruits the DNA translocase ZRANB3 to catalyze fork reversal [PMID:33321094, PMID:37036693]. Biallelic RFWD3 mutations that disrupt RPA binding and chromatin recruitment cause Fanconi anemia complementation group W, with affected cells showing defective HR, ICL hypersensitivity, and chromosomal breakage [PMID:28575657, PMID:28691929]. Beyond the replication-stress core, RFWD3 cooperates with Mdm2 to stabilize p53 in the late DNA-damage response PMID:20173098, and is itself controlled by UHRF1-mediated ubiquitination in a negative-feedback circuit with RAD51 PMID:40940676.

Affinage mechanism profile (Affinage's own GO/Reactome grounding)

molecular_activity: GO:0016874 ligase activity, GO:0140096 catalytic activity, acting on a protein
localization: GO:0005634 nucleus, GO:0000228 nuclear chromosome
pathway (Reactome): R-HSA-73894 DNA Repair, R-HSA-69306 DNA Replication, R-HSA-1643685 Disease, R-HSA-392499 Metabolism of proteins
partners: RPA2, RAD51, PCNA, MDM2, ZRANB3, UHRF1, TREX1
complexes: (none)

Dated findings (citation-anchored)

Year	Confidence	Finding	PMIDs	Journal
2010	Medium	RFWD3 forms a complex with Mdm2 and p53, synergistically ubiquitinates p53 in vitro, and is required to stabilize p53 in the late response to DNA damage. RFWD3-Mdm2 complex restricts polyubiquitination of p53 by Mdm2 alone. RFWD3 is phosphorylated by ATM/ATR kinases, and phosphorylation mutants fail to stimulate p53 ubiquitination.	PMID:20173098	Proceedings of the National Academy of Sciences of the United States of America
2011	Medium	RFWD3 directly interacts with RPA (specifically RPA2 subunit) and is recruited to sites of DNA damage in a RPA-dependent manner. The C-terminus of RFWD3 (coiled-coil and WD40 domains) is necessary for RPA binding. Loss of RFWD3 results in persistent γH2AX and RAD51 foci in damaged cells.	PMID:21558276	The Journal of biological chemistry
2011	Medium	RFWD3 directly interacts with RPA2 using purified proteins, is recruited to stalled replication forks, co-localizes with RPA2 under replication stress, and is required for ATR-dependent Chk1 activation. Deletion of the RPA2-binding region on RFWD3 impairs its localization to stalled forks and decreases Chk1 activation.	PMID:21504906	The Journal of biological chemistry
2015	High	RFWD3 mediates multi-site ubiquitination of the entire RPA complex upon replication fork stalling. Ubiquitination occurs on chromatin at sites outside the DNA binding channel, does not cause proteasomal degradation, and increases under fork collapse conditions. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination at stalled replication forks.	PMID:26474068	Molecular cell
2017	High	RFWD3 mutations cause Fanconi anemia (FANCW). An I639K substitution in the WD40 repeats abolishes interaction of RFWD3 with RPA, preventing RFWD3 recruitment to ICL-stalled replication forks. Single point mutations in RPA32 that abolish RFWD3 interaction also inhibit ICL repair. Unloading of RPA from ICL-induced sites is perturbed in RFWD3-deficient cells.	PMID:28575657	Molecular cell
2017	High	RFWD3 is the FA gene FANCW. Patient-derived mutations in RFWD3 impair its relocation to chromatin and physical interaction with RPA. RFWD3-mutant cells show defective HR, increased ICL sensitivity, G2 arrest, and chromosomal breakage. Rfwd3 knockout mice show increased embryonic lethality, subfertility, and gonadal atrophy.	PMID:28691929	The Journal of clinical investigation
2017	High	RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM is required for this ubiquitination activity in vivo. RFWD3 promotes VCP/p97-mediated protein dynamics and subsequent degradation of both RPA and RAD51, thereby removing them from DNA damage sites. MMC-induced chromatin loading of MCM8 and RAD54 is defective in RFWD3-inactivated cells or cells expressing ubiquitination-deficient RAD51, indicating RAD51 ubiquitination is needed for late-phase HR.	PMID:28575658	Molecular cell
2018	High	RFWD3 localizes at replication forks in unperturbed cells and associates with PCNA via a PIP motif. PCNA association is critical for RFWD3 stability. Cells lacking RFWD3 show slower fork progression, prolonged S phase, and increased loading of replication fork components. The S-phase defect is rescued by WT RFWD3 but not by a PIP mutant. RFWD3-PCNA interaction enables polyubiquitination of RPA for proper DNA replication.	PMID:30530694	Proceedings of the National Academy of Sciences of the United States of America
2020	High	RFWD3 promotes ubiquitylation of proteins on ssDNA at stalled forks, which is required for PCNA ubiquitylation. Absence of RFWD3 leads to defective recruitment of key repair and signaling factors to damaged chromatin, inhibits PCNA ubiquitylation, and drastically impairs translesion DNA synthesis (TLS) across different DNA lesions.	PMID:33321094	Molecular cell
2020	Medium	In BRCA2-deficient cells, stalled forks accumulate phosphorylated and ubiquitinated RPA (ubq-pRPA); the ubiquitination is mediated by RFWD3 and depends on SMARCAL1-dependent fork reversal. Co-depletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity in BRCA2-deficient cells.	PMID:32391871	The Journal of cell biology
2020	Low	The E3 ligase activity of RFWD3 is required for stabilization of the Origin Recognition Complex (ORC) and ORC-Associated protein (ORCA/LRWD1) in cells expressing wild-type p53. RFWD3 associates with ORC/ORCA (Co-IP), and depletion of RFWD3 reduces ORC/ORCA protein levels. Overexpression of ORC/ORCA leads to stabilization of RFWD3.	PMID:33044890	Cell cycle (Georgetown, Tex.)
2020	Medium	The Drosophila ortholog of RFWD3 (Mus302) functions independently of RAD51 during DNA repair, and evidence does not support a role for Mus302 in repair of collapsed replication forks or DSBs through HR — in contrast to the human protein. The N-terminal third of RFWD3 is present only in mammals, suggesting this domain underlies the acquisition of HR-related functions in mammals.	PMID:31900333	G3 (Bethesda, Md.)
2022	Medium	RFWD3 contributes to PCNA modification-dependent DNA damage tolerance independently of both the Fanconi anemia pathway and DNA polymerase η. RFWD3's role in cellular survival after UV irradiation is dependent on PCNA modifications at K164.	PMID:35905994	Life science alliance
2023	High	RFWD3 promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. RFWD3 stimulates fork remodeling (reversal) in a ZRANB3-epistatic manner. RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing the mechanism for ZRANB3 recruitment. Inactivation of RFWD3 in BRCA2-deficient cells rescues fork degradation analogously to ZRANB3 inactivation.	PMID:37036693	The Journal of cell biology
2025	Medium	UHRF1 acts as an E3 ubiquitin ligase for RFWD3, ubiquitinating and destabilizing it to prevent premature RAD51 removal during HR. RAD51 in turn protects RFWD3 from UHRF1, establishing a negative feedback circuit. Phosphorylation of UHRF1 (regulated by phosphatase PP4) controls its E3 activity toward RFWD3.	PMID:40940676	Advanced science (Weinheim, Baden-Wurttemberg, Germany)
2025	Medium	RFWD3 competes with E3 ligase TRIM24 to stabilize TREX1 (a cytosolic dsDNA degrader) by sequestering it from TRIM24-mediated proteasomal degradation. RFWD3 inhibition increases intracellular dsDNA, activates STING-IFN signaling, decreases MDSCs, and enhances PD-L1 blockade efficacy. RFWD3 was identified as a TREX1-interacting protein by mass spectrometry.	PMID:41117130	Advanced science (Weinheim, Baden-Wurttemberg, Germany)
2025	Medium	SLFN11 antagonizes the RFWD3-PRIMPOL fork restart pathway by disrupting recruitment of both RFWD3 and PRIMPOL to stalled forks. In the absence of SLFN11, fork restart proceeds via RFWD3 and PRIMPOL to facilitate gapped DNA synthesis. SLFN11's suppression of RFWD3/PRIMPOL recruitment requires a functional ATPase domain and persistent fork localization, but not tRNA hydrolysis or ssDNA binding.	PMID:41372167	Nature communications

Citations

PMID:20173098
PMID:21504906
PMID:21558276
PMID:26474068
PMID:28575657
PMID:28575658
PMID:28691929
PMID:30530694
PMID:31900333
PMID:32391871
PMID:33044890
PMID:33321094
PMID:35905994
PMID:37036693
PMID:40940676
PMID:41117130
PMID:41372167

📚 Additional Documentation

Notes

(RFWD3-notes.md)

RFWD3 (Q6PCD5) review notes

Gene: RFWD3 / RNF201 ; Fanconi anemia complementation group W (FANCW). Human, 774 aa,
chromosome 16. Domains: degenerate RING-type zinc finger (aa 287-331), coiled-coil
(361-413), WD40 β-propeller (repeats ~495-628, crystallized in PDB 6CVZ residues 425-774).
EC 2.3.2.27 (RING-type E3 ligase). ATM/ATR phospho-substrate at Ser46 and Ser63.

Core biology (synthesis)

RFWD3 is a RING-finger E3 ubiquitin ligase. It is recruited via its C-terminal WD40 domain
to the RPA2 subunit of the RPA complex that coats ssDNA at stalled replication forks / sites
of DNA damage. Once localized, RFWD3 polyubiquitinates RPA (all three subunits) and RAD51,
promoting their VCP/p97-dependent removal/turnover from DNA damage sites so that homologous
recombination (HR) and interstrand crosslink (ICL) repair can progress. It also promotes
ubiquitination of proteins on ssDNA to drive PCNA ubiquitination and translesion synthesis
(TLS). Biallelic RFWD3 mutations cause Fanconi anemia group W (FANCW). A secondary, less
central function is stabilization of p53 in the late DNA-damage response via an RFWD3-MDM2
complex (G1/S checkpoint).

Key provenance

Catalytic activity / MF

PMID:21504906 — first in-vivo characterization.
PMID:26474068
UniProt: EC=2.3.2.27, RING-type (degenerate). Catalytic Cys315 (C315A abolishes activity).

RPA / RAD51 ubiquitination, HR, fork restart

RPA recruitment via WD40 / ICL repair / FANCW

Recruitment to stalled forks / RPA2 binding / checkpoint

PMID:21504906
PMID:21504906
PMID:21504906
[PMID:21558276 title] RFWD3 associates with RPA and facilitates RPA-mediated DNA damage response; up-regulated in S-G2; partially at PML bodies (UniProt subcell).

Translesion synthesis

p53 / MDM2 (secondary role)

Annotation decisions summary

Core MF = RING E3 ubiquitin ligase activity (GO:0061630); process = ICL repair (GO:0036297),
HR (GO:0000724), protein ubiquitination (GO:0016567); locations = nucleus (GO:0005634),
site of DNA damage (GO:0090734).
protein binding (GO:0005515) IPI annotations: all MARK_AS_OVER_ANNOTATED (uninformative;
the biologically meaningful partners RPA2, RAD51, MDM2, p53, UBE2N are captured by specific
terms or the core function).
p53-axis terms (p53 binding, MDM2/MDM4 binding, G1 checkpoint, response to IR) =
KEEP_AS_NON_CORE (secondary, single-group evidence).
cytoplasm / cytosol / PML body = KEEP_AS_NON_CORE (undamaged-cell pool, not the functional site).

📄 View Raw YAML

id: Q6PCD5
gene_symbol: RFWD3
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  RFWD3 (RING finger and WD repeat domain-containing protein 3; also RNF201; Fanconi
  anemia complementation group W, FANCW) is a nuclear RING-type E3 ubiquitin-protein
  ligase (EC 2.3.2.27). It combines an N-terminal region bearing ATM/ATR
  phosphorylation sites (Ser46, Ser63), a degenerate RING zinc finger that provides
  the catalytic ligase activity (active-site Cys315), a coiled-coil, and a C-terminal
  WD40 β-propeller that mediates protein-protein interactions. RFWD3 is recruited,
  through its WD40 domain binding the RPA32 (RPA2) subunit of the single-stranded-DNA-binding
  replication protein A (RPA) complex, to stalled replication forks and sites of DNA
  damage. There it polyubiquitinates RPA (all three subunits) and RAD51, promoting
  their VCP/p97-dependent turnover and timely removal from DNA damage sites so that the
  RPA-to-RAD51 exchange, homologous recombination and interstrand crosslink (ICL) repair
  can proceed. RFWD3 also promotes ubiquitination of proteins on single-stranded DNA,
  driving PCNA ubiquitination and translesion DNA synthesis. It additionally participates
  in replication-checkpoint signaling (ATR-dependent CHK1 activation) and, as a secondary
  activity, cooperates with MDM2 to ubiquitinate and stabilize p53 in the late DNA-damage
  response, contributing to the G1 checkpoint. Biallelic loss-of-function mutations cause
  Fanconi anemia, and Rfwd3-knockout mice show embryonic lethality, subfertility and
  reduced lifespan.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000003
  title: Gene Ontology annotation based on Enzyme Commission mapping
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:19549727
  title: Analysis of the human E2 ubiquitin conjugating enzyme protein interaction
    network.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      High-throughput Y2H map of E2 conjugating enzymes; supports an RFWD3-UBE2N
      (UBC13) interaction consistent with RFWD3 using K63-forming E2 machinery, but
      is a systematic interaction screen, not a functional study of RFWD3.
- id: PMID:20173098
  title: RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in
    response to DNA damage.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Establishes the secondary RFWD3-MDM2-p53 axis; RFWD3 forms a complex with MDM2
      and p53, synergistically ubiquitinates p53, and stabilizes it late after IR in
      an ATM/ATR-phosphorylation-dependent manner. Single-group work; abstract-only
      in cache.
- id: PMID:21504906
  title: E3 ligase RFWD3 participates in replication checkpoint control.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Identifies RFWD3 as a direct RPA2-interacting protein recruited to stalled forks
      and required for ATR-dependent CHK1 activation. Full text available; verified.
- id: PMID:21558276
  title: RING finger and WD repeat domain 3 (RFWD3) associates with replication protein
    A (RPA) and facilitates RPA-mediated DNA damage response.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Companion study confirming RFWD3-RPA association, S-G2 up-regulation, partial
      PML-body localization, and a role in the RPA-mediated DNA damage response.
- id: PMID:24126761
  title: hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance
    of genome integrity.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      Primarily a PRIMPOL study; the RFWD3 annotation derives from a detected
      RFWD3-RPA2 (P15927) interaction, contextual rather than an RFWD3 functional
      characterization.
- id: PMID:25260751
  title: The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      MEKK1/TAB1 study; RFWD3 annotation derives from a detected RFWD3-UBE2N (P61088)
      interaction, not an RFWD3-focused experiment.
- id: PMID:26474068
  title: RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication
    Forks.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Landmark paper: RFWD3 mediates RPA ubiquitination (non-degradative), required
      for fork restart and HR at stalled forks. Full text verified.
- id: PMID:28575657
  title: RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand
    Crosslink Repair and Human Health.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Demonstrates that WD40-mediated RPA binding recruits RFWD3 to ICLs and that the
      FA patient I639K mutation abolishes this; ICL repair is a major RFWD3 function.
      Full text verified.
- id: PMID:28575658
  title: RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51
    from DNA Damage Sites to Facilitate Homologous Recombination.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Identifies RAD51 as a second RFWD3 substrate; RFWD3 polyubiquitinates RPA and
      RAD51 (ATR/ATM-phosphorylation-dependent) driving VCP/p97-mediated removal to
      enable late-phase HR. Abstract-only in cache but definitive for the core function.
- id: PMID:28691929
  title: Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Defines RFWD3 as Fanconi anemia gene FANCW via a patient with biallelic
      mutations; HR disrupted by impaired chromatin relocation and defective RPA
      interaction; supported by a knockout mouse. Full text verified.
- id: PMID:30530694
  title: PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is
    essential for DNA replication.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      RFWD3-focused functional study (full text available). Shows that in unperturbed
      cells RFWD3 localizes at replication forks and binds PCNA via a PIP motif; this
      association stabilizes RFWD3 and supports normal fork progression / DNA
      replication, with reduced RPA ubiquitination in RFWD3-null cells. Corroborates
      the replication-fork-processing role and adds an unperturbed-fork context beyond
      the stalled-fork core function.
- id: PMID:33321094
  title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Shows RFWD3 promotes ubiquitylation of proteins on ssDNA, required for PCNA
      ubiquitylation and translesion synthesis across diverse lesions.
- id: PMID:37036693
  title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled
    replication forks.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      RFWD3-focused functional study (full text available). RFWD3 promotes recruitment
      of the DNA translocase ZRANB3 to stalled forks and stimulates fork remodeling
      (reversal) in a ZRANB3-epistatic manner, via RFWD3-dependent PCNA ubiquitination.
      Corroborates and mechanistically extends the replication-fork-processing role.
- id: PMID:40205054
  title: Multimodal cell maps as a foundation for structural and functional genomics.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      Systems-level multimodal interaction/localization mapping; the RFWD3 annotation
      is a detected RFWD3-RPA2 (P15927) interaction, contextual not functional.
existing_annotations:
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetically inferred RING-type ubiquitin ligase activity. This is the core
      molecular function of RFWD3 and is strongly corroborated by direct biochemistry.
    action: ACCEPT
    reason: >-
      RFWD3 is a RING-finger E3 ligase (EC 2.3.2.27) with catalytic Cys315; multiple
      IDA studies demonstrate ligase activity toward RPA, RAD51 and p53. The IBA is at
      the correct level of specificity and represents the central evolved function.
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination.
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Phylogenetically inferred involvement in protein ubiquitination, the process
      carried out by the RFWD3 ligase activity.
    action: ACCEPT
    reason: >-
      Directly supported: RFWD3 ubiquitinates RPA, RAD51 and p53 and promotes
      ubiquitination of proteins on ssDNA. Correct and well-supported.
    supported_by:
    - reference_id: PMID:33321094
      supporting_text: the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      Phylogenetically inferred nuclear localization; RFWD3 acts in the nucleus at
      replication forks and DNA damage sites.
    action: ACCEPT
    reason: >-
      RFWD3 functions in the nucleus where it ubiquitinates RPA/RAD51 at chromatin;
      is_active_in nucleus is the correct core location.
    supported_by:
    - reference_id: PMID:28691929
      supporting_text: impaired relocation of mutant RFWD3 to chromatin
- term:
    id: GO:0031297
    label: replication fork processing
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Phylogenetically inferred role at stalled replication forks, consistent with
      RFWD3 promoting fork restart and repair at stalled forks.
    action: ACCEPT
    reason: >-
      RFWD3 is recruited to stalled replication forks and is required for fork restart
      and repair at stalled forks (Elia et al. 2015).
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: RFWD3 is necessary for replication fork restart, normal repair
        kinetics during
- term:
    id: GO:0090734
    label: site of DNA damage
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      Phylogenetically inferred activity at sites of DNA damage; RFWD3 is recruited to
      and functions at DNA damage sites.
    action: ACCEPT
    reason: >-
      RFWD3 translocates to tracks of laser micro-irradiation and to MMC-induced foci
      that co-localize with RPA and gamma-H2AX; this is a core functional location.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: translocates to tracks of laser micro-irradiation and co-localizes
        with the phosphorylated form of histone variant H2AX
- term:
    id: GO:0004842
    label: ubiquitin-protein transferase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro-based electronic annotation of ubiquitin-protein transferase activity,
      a parent/equivalent of the demonstrated RING ligase activity.
    action: ACCEPT
    reason: >-
      Correct: RFWD3 is a ubiquitin transferase (RING E3). Slightly more general than
      GO:0061630 but not wrong; the specific ligase activity is captured by the IDA
      annotations.
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    summary: Electronic annotation of nuclear localization, consistent with experimental data.
    action: ACCEPT
    reason: >-
      Nuclear localization is experimentally established (multiple EXP/IDA annotations);
      the IEA is correct.
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      Electronic subcellular-location annotation of cytoplasm; RFWD3 is found in the
      cytoplasm of undamaged cells before nuclear recruitment upon damage.
    action: KEEP_AS_NON_CORE
    reason: >-
      A cytoplasmic pool is documented in undamaged cells, but the functional site of
      RFWD3 is the nucleus/chromatin; cytoplasmic localization is peripheral to its
      DNA-repair function.
    supported_by:
    - reference_id: PMID:21558276
      supporting_text: RFWD3 associates with replication protein
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: involved_in
  review:
    summary: Electronic annotation of protein ubiquitination, matching experimental evidence.
    action: ACCEPT
    reason: RFWD3 ubiquitinates multiple substrates; the process annotation is correct.
- term:
    id: GO:0016605
    label: PML body
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      Electronic subcellular-location annotation of PML nuclear body, from the reported
      partial association of RFWD3 with PML bodies in undamaged cells.
    action: KEEP_AS_NON_CORE
    reason: >-
      RFWD3 is partially associated with PML nuclear bodies in undamaged cells, but this
      is not the site of its characterized DNA-repair ligase activity.
    supported_by:
    - reference_id: PMID:21558276
      supporting_text: RFWD3 associates with replication protein
- term:
    id: GO:0036297
    label: interstrand cross-link repair
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: involved_in
  review:
    summary: >-
      InterPro-based annotation of interstrand cross-link repair, a core RFWD3 process
      confirmed experimentally.
    action: ACCEPT
    reason: >-
      RFWD3-deficient cells are profoundly hypersensitive to ICL-inducing agents; ICL
      repair is a major RFWD3 function (FANCW).
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: show profound defects in ICL repair
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000003
  qualifier: enables
  review:
    summary: EC-mapping-based annotation of ubiquitin ligase activity (EC 2.3.2.27).
    action: ACCEPT
    reason: >-
      Consistent with the RecName EC 2.3.2.27 and direct biochemical demonstration of
      RING E3 ligase activity.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:19549727
  qualifier: enables
  review:
    summary: >-
      IntAct-curated interaction with the E2 conjugating enzyme UBE2N (UBC13) from a
      systematic E2 interaction-network screen.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      GO:0005515 protein binding is uninformative as a molecular function. The
      biologically relevant interaction (RFWD3 partnering with the K63-forming E2
      UBE2N) is better captured by the ligase activity core function; no specific MF
      term is warranted from this high-throughput screen.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20173098
  qualifier: enables
  review:
    summary: >-
      IntAct-curated interactions with p53 (TP53) and MDM2 from the RFWD3-MDM2-p53
      study.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Generic protein binding is uninformative; the specific and informative
      interactions are separately annotated as p53 binding (GO:0002039) and MDM2/MDM4
      family protein binding (GO:0097371).
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:24126761
  qualifier: enables
  review:
    summary: IntAct-curated RFWD3-RPA2 (P15927) interaction detected in a PRIMPOL study.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Uninformative generic binding term; the functionally important RPA2 interaction
      underlies the core RFWD3 recruitment/ubiquitination function and is captured there.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:25260751
  qualifier: enables
  review:
    summary: IntAct-curated RFWD3-UBE2N (P61088) interaction detected in a MEKK1/TAB1 study.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Generic protein binding is uninformative; the UBE2N (E2) partnership is subsumed
      by the ligase activity annotation.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:28691929
  qualifier: enables
  review:
    summary: IntAct-curated RFWD3-RPA2 (P15927) interaction from the FANCW paper.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Uninformative generic term; the RPA2 interaction that recruits RFWD3 to chromatin
      is central and captured by the core function rather than by GO:0005515.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:40205054
  qualifier: enables
  review:
    summary: >-
      IntAct-curated RFWD3-RPA2 (P15927) interaction from a systems-level multimodal
      cell-mapping study.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Generic protein binding is uninformative; the RPA2 interaction is already
      represented functionally elsewhere.
- term:
    id: GO:0005654
    label: nucleoplasm
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  qualifier: located_in
  review:
    summary: HPA immunofluorescence localization to the nucleoplasm.
    action: ACCEPT
    reason: >-
      Consistent with the established nuclear localization; nucleoplasm is an accurate
      sub-nuclear location.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  qualifier: located_in
  review:
    summary: HPA immunofluorescence localization to the cytosol.
    action: KEEP_AS_NON_CORE
    reason: >-
      A cytosolic pool is consistent with cytoplasmic localization in undamaged cells,
      but is peripheral to the nuclear DNA-repair function.
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: EXP
  original_reference_id: PMID:21504906
  qualifier: located_in
  review:
    summary: >-
      Experimental nuclear localization; RFWD3 is recruited to the nucleus/stalled
      forks upon replication stress.
    action: ACCEPT
    reason: Nuclear localization is well established experimentally.
    supported_by:
    - reference_id: PMID:21504906
      supporting_text: RFWD3 is recruited to stalled replication forks and co-localizes
        with RPA2 in response to replication stress
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: EXP
  original_reference_id: PMID:21558276
  qualifier: located_in
  review:
    summary: Experimental nuclear localization from the RFWD3-RPA association study.
    action: ACCEPT
    reason: Consistent with established nuclear localization.
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: EXP
  original_reference_id: PMID:28575657
  qualifier: located_in
  review:
    summary: >-
      Experimental nuclear localization; GFP-RFWD3 forms nuclear foci/tracks at DNA
      damage sites.
    action: ACCEPT
    reason: Nuclear localization at DNA damage sites is directly demonstrated.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: translocates to tracks of laser micro-irradiation
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: EXP
  original_reference_id: PMID:21558276
  qualifier: located_in
  review:
    summary: >-
      Experimental cytoplasmic localization; RFWD3 is found in the cytoplasm of
      undamaged cells.
    action: KEEP_AS_NON_CORE
    reason: >-
      Real localization in undamaged cells but peripheral to the nuclear repair function.
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: EXP
  original_reference_id: PMID:28691929
  qualifier: located_in
  review:
    summary: Experimental cytoplasmic localization reported in the FANCW study.
    action: KEEP_AS_NON_CORE
    reason: >-
      Cytoplasmic pool documented; the functionally critical relocation is to
      chromatin/nucleus.
- term:
    id: GO:0036297
    label: interstrand cross-link repair
  evidence_type: IDA
  original_reference_id: PMID:33321094
  qualifier: involved_in
  review:
    summary: >-
      Direct demonstration of RFWD3's role in the crosslink/lesion-bypass response;
      RFWD3 promotes ssDNA-protein ubiquitination required for TLS during ICL repair.
    action: ACCEPT
    reason: >-
      RFWD3 is required for translesion synthesis, an integral gap-filling step of ICL
      repair; ICL repair involvement is a core function.
    supported_by:
    - reference_id: PMID:33321094
      supporting_text: TLS across different DNA lesions is drastically impaired
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IDA
  original_reference_id: PMID:20173098
  qualifier: enables
  review:
    summary: >-
      Direct demonstration of RFWD3 E3 ligase activity toward p53 (in the RFWD3-MDM2
      complex).
    action: ACCEPT
    reason: >-
      In vitro experiments show RFWD3 acts as a p53 E3 ligase; supports the core RING
      ligase activity. (The p53 process context is secondary.)
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: RFWD3 is a p53 E3 ubiquitin ligase
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IDA
  original_reference_id: PMID:26474068
  qualifier: enables
  review:
    summary: Direct demonstration that RFWD3 ligase activity ubiquitinates RPA.
    action: ACCEPT
    reason: Core catalytic function; RFWD3 mediates RPA ubiquitination.
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: the E3 ligase RFWD3 is responsible for RPA ubiquitination
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IDA
  original_reference_id: PMID:28575657
  qualifier: enables
  review:
    summary: >-
      Direct demonstration of RFWD3 ligase activity; the recombinant C315A active-site
      mutant is inactive in vitro.
    action: ACCEPT
    reason: >-
      Confirms catalytic RING E3 activity dependent on Cys315; core molecular function.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: which renders the recombinant protein inactive in vitro
- term:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  evidence_type: IDA
  original_reference_id: PMID:28575658
  qualifier: enables
  review:
    summary: >-
      Direct demonstration that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and
      in vivo.
    action: ACCEPT
    reason: Core catalytic function toward the physiological substrates RPA and RAD51.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo
- term:
    id: GO:0000724
    label: double-strand break repair via homologous recombination
  evidence_type: IMP
  original_reference_id: PMID:28691929
  qualifier: involved_in
  review:
    summary: >-
      Loss-of-function (patient/engineered mutations) disrupts homologous recombination,
      establishing RFWD3's role in HR.
    action: ACCEPT
    reason: >-
      HR was disrupted in RFWD3-mutant cells; a core process for RFWD3 (FANCW).
    supported_by:
    - reference_id: PMID:28691929
      supporting_text: HR was disrupted in RFWD3-mutant cells
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IMP
  original_reference_id: PMID:28691929
  qualifier: located_in
  review:
    summary: >-
      Mutation-based evidence that RFWD3 relocation to the nucleus/chromatin is required
      for function.
    action: ACCEPT
    reason: >-
      Nuclear/chromatin relocation is impaired by FA mutations, underscoring the nucleus
      as the functional compartment.
    supported_by:
    - reference_id: PMID:28691929
      supporting_text: impaired relocation of mutant RFWD3 to chromatin
- term:
    id: GO:0000724
    label: double-strand break repair via homologous recombination
  evidence_type: IDA
  original_reference_id: PMID:26474068
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 is required for homologous recombination at stalled replication forks.
    action: ACCEPT
    reason: Core process; RFWD3 stimulates HR at stalled forks.
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: homologous recombination (HR) at stalled replication forks
- term:
    id: GO:0000724
    label: double-strand break repair via homologous recombination
  evidence_type: IDA
  original_reference_id: PMID:28575657
  qualifier: involved_in
  review:
    summary: >-
      RFWD3-deficient cells show HR defects (PARP-inhibitor hypersensitivity, persistent
      RPA in RAD51 foci).
    action: ACCEPT
    reason: >-
      Supports the core HR function; RFWD3 controls RPA/RAD51 dynamics required for HR.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: unloading of RPA from sites of ICL induction is perturbed in
        RFWD3-deficient cells
- term:
    id: GO:0000724
    label: double-strand break repair via homologous recombination
  evidence_type: IDA
  original_reference_id: PMID:28575658
  qualifier: involved_in
  review:
    summary: >-
      RFWD3-mediated removal of RPA and RAD51 facilitates late-phase homologous
      recombination.
    action: ACCEPT
    reason: Core HR process, mechanistically defined via RPA/RAD51 turnover.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: crucial for progression to the late-phase HR and suppression
        of the FA phenotype
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:28575657
  qualifier: enables
  review:
    summary: >-
      IntAct-curated interactions with RPA2 (P15927) and RAD51 (Q06609), the physiological
      RFWD3 substrates/partners.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Generic protein binding is uninformative as an MF; the functionally meaningful
      RPA2 and RAD51 interactions underpin the core ligase/HR function and are captured
      there.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:28575658
  qualifier: enables
  review:
    summary: IntAct-curated RFWD3-RPA2 (P15927) interaction.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Uninformative generic binding term; the RPA2 interaction is functionally
      represented by the core function.
- term:
    id: GO:0006974
    label: DNA damage response
  evidence_type: IDA
  original_reference_id: PMID:26474068
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 acts within the DNA damage response (RPA ubiquitination upon replication
      stress).
    action: ACCEPT
    reason: >-
      Accurate high-level process; RFWD3's ligase activity is part of the DDR. More
      specific processes (HR, ICL repair, fork processing) are also annotated.
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: to profile ubiquitination in the DNA damage response (DDR)
- term:
    id: GO:0006974
    label: DNA damage response
  evidence_type: IDA
  original_reference_id: PMID:28575657
  qualifier: involved_in
  review:
    summary: RFWD3 recruitment and action at DNA damage sites within the DDR.
    action: ACCEPT
    reason: Correct high-level process annotation, consistent with damage-site recruitment.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: translocates to tracks of laser micro-irradiation
- term:
    id: GO:0006974
    label: DNA damage response
  evidence_type: IDA
  original_reference_id: PMID:28575658
  qualifier: involved_in
  review:
    summary: RFWD3 acts in the DDR via ATR/ATM-regulated ubiquitination of RPA and RAD51.
    action: ACCEPT
    reason: Correct high-level DDR process; RFWD3 activity depends on ATR/ATM phosphorylation.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: Phosphorylation by ATR and ATM kinases is required for this activity
        in vivo
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IDA
  original_reference_id: PMID:26474068
  qualifier: involved_in
  review:
    summary: Direct evidence of RFWD3-mediated RPA ubiquitination.
    action: ACCEPT
    reason: Core process; RFWD3 ubiquitinates RPA on chromatin.
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: the E3 ligase RFWD3 is responsible for RPA ubiquitination
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IDA
  original_reference_id: PMID:28575657
  qualifier: involved_in
  review:
    summary: RFWD3 ubiquitinates RPA at sites of ICL-induced fork stalling.
    action: ACCEPT
    reason: Core process; RFWD3 ubiquitylates RPA.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: interacts with and ubiquitylates replication protein A (RPA)
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IDA
  original_reference_id: PMID:28575658
  qualifier: involved_in
  review:
    summary: RFWD3 polyubiquitinates RPA and RAD51.
    action: ACCEPT
    reason: Core process, defined toward physiological substrates.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo
- term:
    id: GO:0031297
    label: replication fork processing
  evidence_type: IDA
  original_reference_id: PMID:26474068
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 promotes restart and remodeling of stalled replication forks and supports
      normal fork progression.
    action: ACCEPT
    reason: >-
      Direct evidence that RFWD3 is necessary for fork restart and repair at stalled
      forks (Elia et al. 2015); further corroborated by its role in normal fork
      progression via PCNA binding (Lin et al. 2018) and in ZRANB3-dependent fork
      remodeling/reversal (Moore et al. 2023).
    supported_by:
    - reference_id: PMID:26474068
      supporting_text: we found that depletion of RFWD3 decreased fork restart
    - reference_id: PMID:30530694
      supporting_text: in unperturbed human cells, RFWD3 localizes at replication forks
        and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting
        protein (PIP) motif
    - reference_id: PMID:37036693
      supporting_text: RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner
- term:
    id: GO:0036297
    label: interstrand cross-link repair
  evidence_type: IMP
  original_reference_id: PMID:28575657
  qualifier: involved_in
  review:
    summary: >-
      RFWD3-hypomorphic cells are profoundly hypersensitive to ICL-inducing agents,
      demonstrating an ICL-repair role.
    action: ACCEPT
    reason: >-
      Loss-of-function evidence establishes ICL repair as a major RFWD3 function (core).
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: argues strongly that ICL repair is a major function of RFWD3
- term:
    id: GO:0036297
    label: interstrand cross-link repair
  evidence_type: IMP
  original_reference_id: PMID:28575658
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 inactivation impairs ICL repair; suppression of the FA phenotype depends on
      RFWD3-mediated RPA/RAD51 removal.
    action: ACCEPT
    reason: Core ICL-repair process consistent with FANCW identity.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: suppression of the FA phenotype
- term:
    id: GO:0090734
    label: site of DNA damage
  evidence_type: IDA
  original_reference_id: PMID:28575657
  qualifier: located_in
  review:
    summary: RFWD3 localizes to sites of ICL/DNA damage (laser tracks, MMC foci).
    action: ACCEPT
    reason: Core functional location, directly imaged.
    supported_by:
    - reference_id: PMID:28575657
      supporting_text: translocates to tracks of laser micro-irradiation
- term:
    id: GO:0090734
    label: site of DNA damage
  evidence_type: IDA
  original_reference_id: PMID:28575658
  qualifier: located_in
  review:
    summary: RFWD3 acts at DNA damage sites to remove RPA and RAD51.
    action: ACCEPT
    reason: >-
      Consistent with damage-site localization and function; core functional location.
    supported_by:
    - reference_id: PMID:28575658
      supporting_text: timely removal of RPA and RAD51 from DNA damage sites
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:21504906
  qualifier: enables
  review:
    summary: >-
      IntAct-curated direct RFWD3-RPA2 (P15927) interaction, shown with purified
      proteins.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Generic protein binding is uninformative; the direct RPA2 interaction that
      recruits RFWD3 to stalled forks is central and represented by the core function.
- term:
    id: GO:0035861
    label: site of double-strand break
  evidence_type: IDA
  original_reference_id: PMID:21504906
  qualifier: colocalizes_with
  review:
    summary: >-
      RFWD3 co-localizes with RPA2 at stalled forks/damage sites in response to
      replication stress.
    action: ACCEPT
    reason: >-
      Accurate colocalization annotation consistent with recruitment to damage sites;
      complements the site of DNA damage annotations.
    supported_by:
    - reference_id: PMID:21504906
      supporting_text: RFWD3 is recruited to stalled replication forks and co-localizes
        with RPA2 in response to replication stress
- term:
    id: GO:2000001
    label: regulation of DNA damage checkpoint
  evidence_type: IMP
  original_reference_id: PMID:21504906
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 depletion decreases ATR-dependent CHK1 activation after replication stress,
      implicating it in replication-checkpoint control.
    action: KEEP_AS_NON_CORE
    reason: >-
      A genuine but secondary/context-dependent role; the effect on CHK1 activation is
      cell-type dependent and is distinct from the core RPA/RAD51-ubiquitination repair
      function.
    supported_by:
    - reference_id: PMID:21504906
      supporting_text: RFWD3 is important for ATR-dependent Chk1 activation in response
        to replication stress
- term:
    id: GO:0002039
    label: p53 binding
  evidence_type: IPI
  original_reference_id: PMID:20173098
  qualifier: enables
  review:
    summary: >-
      Direct interaction with p53 (TP53) within the RFWD3-MDM2-p53 complex that regulates
      p53 stability.
    action: KEEP_AS_NON_CORE
    reason: >-
      An informative, specific binding (unlike generic protein binding), but it underlies
      the secondary p53-stabilization role rather than the core DNA-repair ligase function.
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: forms a complex with Mdm2 and p53 to synergistically ubiquitinate
        p53
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IDA
  original_reference_id: PMID:20173098
  qualifier: located_in
  review:
    summary: Experimental nuclear localization in the p53-regulation study.
    action: ACCEPT
    reason: Consistent with established nuclear localization.
- term:
    id: GO:0010212
    label: response to ionizing radiation
  evidence_type: IDA
  original_reference_id: PMID:20173098
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 is phosphorylated and stabilizes p53 in the late response to ionizing
      radiation.
    action: KEEP_AS_NON_CORE
    reason: >-
      A real damage-response context, but tied to the secondary p53 role; the core RFWD3
      function is better described by HR/ICL-repair processes.
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: p53 stabilization in the late phase after ionizing radiation
        correlates with active ubiquitination
- term:
    id: GO:0016567
    label: protein ubiquitination
  evidence_type: IDA
  original_reference_id: PMID:20173098
  qualifier: involved_in
  review:
    summary: RFWD3 ubiquitinates p53 (with MDM2) in the late DNA-damage response.
    action: ACCEPT
    reason: >-
      A genuine protein-ubiquitination event catalyzed by RFWD3; correct process term,
      here in the secondary p53 context.
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: forms a complex with Mdm2 and p53 to synergistically ubiquitinate
        p53
- term:
    id: GO:0031571
    label: mitotic G1 DNA damage checkpoint signaling
  evidence_type: IMP
  original_reference_id: PMID:20173098
  qualifier: involved_in
  review:
    summary: >-
      RFWD3 positively regulates p53 stability when the G1 cell-cycle checkpoint is
      activated.
    action: KEEP_AS_NON_CORE
    reason: >-
      Secondary function via p53/MDM2; distinct from the core replication-associated
      repair activity and supported by a single group.
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: a positive regulator of p53 stability when the G(1) cell cycle
        checkpoint is activated
- term:
    id: GO:0097371
    label: MDM2/MDM4 family protein binding
  evidence_type: IPI
  original_reference_id: PMID:20173098
  qualifier: enables
  review:
    summary: Direct interaction with MDM2 in the p53-regulatory RFWD3-MDM2 complex.
    action: KEEP_AS_NON_CORE
    reason: >-
      Informative specific binding supporting the secondary p53-stabilization role, not
      the core DNA-repair ligase function.
    supported_by:
    - reference_id: PMID:20173098
      supporting_text: RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53
core_functions:
- description: >-
    RING-type E3 ubiquitin-protein ligase that is recruited via its C-terminal WD40
    domain to RPA-coated single-stranded DNA at stalled replication forks and sites of
    DNA damage, where (following ATM/ATR phosphorylation) it polyubiquitinates the RPA
    complex and RAD51 to drive their VCP/p97-dependent turnover and timely removal,
    enabling homologous recombination and interstrand crosslink repair to progress.
  supported_by:
  - reference_id: PMID:26474068
    supporting_text: the E3 ligase RFWD3 is responsible for RPA ubiquitination
  - reference_id: PMID:28575658
    supporting_text: facilitates timely removal
  - reference_id: PMID:28575657
    supporting_text: interacts with and ubiquitylates replication protein A (RPA)
  molecular_function:
    id: GO:0061630
    label: ubiquitin protein ligase activity
  directly_involved_in:
  - id: GO:0036297
    label: interstrand cross-link repair
  - id: GO:0000724
    label: double-strand break repair via homologous recombination
  - id: GO:0016567
    label: protein ubiquitination
  locations:
  - id: GO:0090734
    label: site of DNA damage
  - id: GO:0005634
    label: nucleus

RFWD3

Gene Description