HGNC Approved Gene Symbol: RFWD3
Cytogenetic location: 16q23.1 Genomic coordinates (GRCh38): 16:74,621,399-74,666,877 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 16q23.1 | ?Fanconi anemia, complementation group W | 617784 | Autosomal recessive | 3 |
RFWD3 is an E3 ubiquitin ligase that participates in DNA damage response in association with the replication protein A complex (see RPA1, 179835) and by stabilizing p53 (TP53; 191170) (summary by Liu et al., 2011).
Fu et al. (2010) cloned full-length human RFWD3 from a HeLa cell cDNA library. The deduced 774-amino acid protein contains an N-terminal SQ-rich region, followed by a RING domain, a coiled-coil domain, and a C-terminal WD40 domain.
Liu et al. (2011) found that the SQ-rich N-terminal domain of RFWD3, called an SSQ domain, is made up of 3 near perfect 17-amino acid repeats that includes the motif SSQ.
In unstressed cells, p53 is polyubiquitinated by the E3 ubiquitin ligase MDM2 (164785) and is targeted for proteolytic degradation. However, in response to DNA damage, p53 is stabilized and activates transcriptional programs essential for cell cycle arrest and apoptosis. Fu et al. (2010) found that RFWD3 stabilized p53 in response to DNA damage. RFWD3 formed a complex with MDM2 and p53 and synergized with MDM2 for monoubiquitination of p53, but the interaction restricted MDM2 from destabilizing p53 by extending the polyubiquitin chain. RFWD3 was also phosphorylated by the checkpoint kinases ATM (607585) and ATR (601215), which further enhanced p53 monoubiquitination after DNA damage. Fu et al. (2010) concluded that RFWD3 is an E3 ubiquitin ligase that stabilizes p53 when the G1 cell cycle checkpoint is activated.
By tandem affinity purification of 293T cells, followed by mass spectrometric analysis, Gong and Chen (2011) found that RFWD3 copurified with the RPA complex, which binds and stabilizes single-stranded DNA at sites of DNA damage. In vitro pull-down assays revealed that RFWD3 specifically interacted with RPA2 (179836), but not with RPA1 or RPA3 (179837). Use of truncation and internal deletion mutants revealed that the coiled-coil domain of RFWD3 specifically interacted with a domain near the C terminus of RPA2. Immunohistochemical analysis revealed that RFWD3 colocalized with RPA2 at hydroxyurea-induced foci of DNA damage. Knockdown of RFWD3 via small interfering RNA caused failure to phosphorylate the checkpoint protein CHK1 (CHEK1; 603078) following DNA damage. Failure to activate CHK1 was also observed with a RFWD3 mutant unable to interact with RPA2 and with a RFWD3 mutant lacking the E3 ubiquitin ligase RING domain. Loss of RFWD3 caused hypersensitivity to hydroxyurea. Gong and Chen (2011) concluded that the E3 ubiquitin ligase activity of RFWD3 is critical for replication checkpoint.
Liu et al. (2011) found that RFWD3 partly localized to PML (102578) nuclear storage bodies during S/G2 phase in normal growing human cells and translocated to sites of DNA damage in an RPA-dependent manner. RFWD3 directly interacted with RPA2 via its coiled-coil domain and WD40 repeats and required this interaction for accumulation at sites of DNA damage. Knockdown of either RFWD3 or RPA2 attenuated phosphorylation of both proteins. Loss of RFWD3 delayed RAD51 (179617) focus formation in response to replication block and correlated with persistence of RAD51 and phosphorylated H2AX (601772) foci, indicating defective DNA repair.
Hartz (2011) mapped the RFWD3 gene to chromosome 16q23.1 based on an alignment of the RFWD3 sequence (GenBank AK001382) with the genomic sequence (GRCh37).
In a 12-year-old German girl (patient 1143), born of unrelated parents, with Fanconi anemia complementation group W (FANCW; 617784), Knies et al. (2017) identified compound heterozygous mutations in the RFWD3 gene (c.205_206dupCC, 614151.0001 and I639K, 614151.0002). Patient cells showed increased chromosomal breakage, reduced survival, and cell cycle arrest in G2 following exposure to mitomycin C (MMC) and additional DNA cross-linking agents, and these defects could be restored by expression of wildtype RFWD3. The specific results suggested a defect in BRCA2 (600185)-dependent homologous recombination (HR). In vitro studies of the I639K variant indicated that it had less relocalization to the nucleus and to chromatin compared to wildtype, had disrupted physical interaction with RPA proteins (see, e.g., RPA2, 179836), and caused impaired HR in transduced cells. Three different cellular models with generation of RFWD3 mutants recapitulated the defects observed in patient cells, indicating that RFWD3 normally promotes HR induced by DNA interstrand cross-links. Knies et al. (2017) concluded that RFWD3 is positioned late in the FA/BRCA pathway, downstream of BRCA2/FANCD1.
Knies et al. (2017) found that Rfwd3-null mice were viable and did not show overt phenotypic abnormalities, although there was some evidence for increased embryonic lethality, earlier death, and subfertility, associated with testicular and ovarian atrophy in mutant mice. Mutant mouse embryonic fibroblasts were hypersensitive to DNA cross-linking agents and showed increased chromosomal breakage compared to controls.
In a 12-year-old German girl (patient 1143), born of unrelated parents, with Fanconi anemia complementation group W (FANCW; 617784), Knies et al. (2017) identified compound heterozygous mutations in the RFWD3 gene: a 2-bp duplication (c.205_206dupCC, NM_018124.3), resulting in a frameshift and premature termination (Leu69ProfsTer12) in the N-terminal region, and a c.1916T-A transversion, resulting in an ile639-to-lys (I639K; 614151.0002) substitution at a highly conserved residue in the WD40 domain, which is responsible for RPA2 (179836) binding. Neither mutation was found in the 1000 Genomes Project or the ExAC database. The c.205_206dupCC mutation was predicted to result in nonsense-mediated mRNA decay and a functionally null allele, since a truncated protein was not detected in patient cells. Patient cells showed increased chromosomal breakage following exposure to mitomycin C (MMC) and additional disruptive agents, and these defects could be restored by expression of wildtype RFWD3. The specific results suggested a defect in BRCA2 (600185)-dependent homologous recombination (HR). In vitro studies of the I639K variant indicated that it had less relocalization to the nucleus and to chromatin compared to wildtype as well as impaired interaction with RPA2, resulting in impaired HR in transduced cells.
For discussion of the c.1916T-A transversion (c.1916T-A, NM_018124.3) in the RFWD3 gene, resulting in an ile639-to-lys (I639K) substitution, that was found in compound heterozygous state in a patient with Fanconi anemia complementation group W (FANCW; 617784) by Knies et al. (2017), see 614151.0001.
Fu, X., Yucer, N., Liu, S., Li, M., Yi, P., Mu, J.-J., Yang, T., Chu, J., Jung, S. Y., O'Malley, B. W., Gu, W., Qin, J., Wang, Y. RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage. Proc. Nat. Acad. Sci. 107: 4579-4584, 2010. [PubMed: 20173098] [Full Text: https://doi.org/10.1073/pnas.0912094107]
Gong, Z., Chen, J. E3 ligase RFWD3 participates in replication checkpoint control. J. Biol. Chem. 286: 22308-22313, 2011. [PubMed: 21504906] [Full Text: https://doi.org/10.1074/jbc.M111.222869]
Hartz, P. A. Personal Communication. Baltimore, Md. 8/5/2011.
Knies, K., Inano, S., Ramirez, M. J., Ishiai, M., Surralles, J., Takata, M., Schindler, D. Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia. J. Clin. Invest. 127: 3013-3027, 2017. [PubMed: 28691929] [Full Text: https://doi.org/10.1172/JCI92069]
Liu, S., Chu, J., Yucer, N., Leng, M., Wang, S.-Y., Chen, B. P. C., Hittelman, W. N., Wang, Y. RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response. J. Biol. Chem. 286: 22314-22322, 2011. [PubMed: 21558276] [Full Text: https://doi.org/10.1074/jbc.M111.222802]