Alternative titles; symbols
HGNC Approved Gene Symbol: SLX4
Cytogenetic location: 16p13.3 Genomic coordinates (GRCh38): 16:3,581,181-3,611,606 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 16p13.3 | Fanconi anemia, complementation group P | 613951 | Autosomal recessive | 3 |
SLX4 functions as a critical scaffold element for the assembly of a multiprotein complex containing enzymes involved in DNA maintenance and repair (Svendsen et al., 2009).
By sequencing clones obtained from size-fractionated fetal brain cDNA libraries, Nagase et al. (2001, 2001) cloned the partial cDNAs KIAA1987 and KIAA1784, which represent the 5-prime and 3-prime ends of SLX4, respectively. Both cDNAs contain repetitive elements in their UTRs. RT-PCR ELISA detected low to moderate expression in all adult and fetal tissues and specific adult brain regions examined.
By searching for sequences similar to fungal Slx4 proteins and Drosophila Mus312, Fekairi et al. (2009) identified human SLX4. The deduced 1,834-amino acid protein contains 2 N-terminal C2HC-type zinc fingers, followed by a putative protein-protein interaction motif, a central BTB/POZ domain, a SAP motif, and a conserved C-terminal domain.
By analyzing proteins that purified with epitope-tagged SLX4 from human embryonic kidney cells, Svendsen et al. (2009) showed that SLX4 associated with the heterodimeric DNA flap endonucleases ERCC4 (133520)-ERCC1 (126380) and MUS81 (606591)-EME1 (610885) and the human ortholog of the yeast Slx1 endonuclease. In humans, 2 genes, GIYD1 (SLX1A; 615822) and GIYD2 (SLX1B; 615823), encode identical SLX1 proteins. SLX4 also associated with the telomere-interacting protein TERF2 (602027) and its partner TERF2IP (605061), the protein kinase PLK1 (602098), the MSH2 (609309)-MSH3 (600887) mismatch repair complex, and C20ORF94 (SLX4IP; 615958). Similar complexes were detected in other human cell lines examined. Domain analysis showed that MSH2-MSH3, ERCC4-ERCC1, and C20ORF94 associated with the N-terminal region of SLX4, while TERF2-TERF2IP, MUS81-EME1, and PLK1 bound the C-terminal region of SLX4. SLX1 associated with the extreme C terminus of SLX4. The SLX4 complex was recruited to sites of DNA damage and repaired 3-prime flap, 5-prime flap, and replication fork structures, consistent with the activities of the SLX4-associated DNA repair factors. The SLX1-SLX4 module specifically promoted symmetrical cleavage of static and migrating Holliday junctions. PLK1 phosphorylated SLX4 in situ, suggesting that phosphorylation of SLX4 regulates DNA repair by the SLX4 complex. Svendsen et al. (2009) concluded that the SLX4 complex is required for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure.
Using yeast 2-hybrid assays, Fekairi et al. (2009) showed that human SLX4 interacted with SLX1. Mutation analysis showed that the C-terminal domain of SLX4 was required for the interaction. The SLX1-SLX4 duplex functioned as a 5-prime flap endonuclease against a DNA stem loop substrate, and it resolved Holliday junctions into linear duplex products in the presence of Mn(2+). SLX1-SLX4 duplex activity was dependent on SLX1. However, SLX4, but not SLX1, was required for interstrand crosslink repair, suggesting that SLX4 functions with other endonucleases to repair interstrand crosslinks.
Wechsler et al. (2011) used Bloom syndrome (210900) cells, in which the BLM gene (604610) is inactive, to analyze human cells compromised for the known Holliday junction dissolution/resolution pathways. Wechsler et al. (2011) showed that depletion of MUS81 and GEN1 (612449), or SLX4 and GEN1, from Bloom syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Wechsler et al. (2011) concluded that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, Wechsler et al. (2011) showed that depletion of MUS81 or SLX4 reduces the high frequency of sister chromatid exchanges in Bloom syndrome cells, indicating that MUS81 and SLX4 promote sister chromatid exchange formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom syndrome.
Hartz (2010) mapped the SLX4 gene to chromosome 16p13.3 based on an alignment of the SLX4 sequence (GenBank AK095411) with the genomic sequence (GRCh37).
In a Dutch boy, born of consanguineous parents, with Fanconi anemia of complementation group P (FANCP; 613951), Stoepker et al. (2011) identified a homozygous truncating mutation in the SLX4 gene (613278.0001). The gene was chosen for study because of its known function as a scaffold protein in the DNA repair pathway. Stoepker et al. (2011) identified compound heterozygous mutations in the SLX4 gene (613278.0002 and 613278.0003) in 3 German sibs with a milder FANCP phenotype.
Kim et al. (2011) identified biallelic mutations in the SLX4 gene (613278.0004-613278.0006) in 2 unrelated patients with FANCP.
Crossan et al. (2011) found that Slx4-null mice recapitulated the features of Fanconi anemia in humans. Slx4-null mice were born at submendelian ratios and had greatly reduced fertility due to gonad dysfunction. Ovaries showed absence of oocytes, and testes showed progressive failure of spermatogenesis. Many mutant mice died soon after birth, and the survivors showed poor growth, with domed skulls and ocular anomalies. Mutant mice also showed blood cytopenia, indicating hematologic dysfunction. Cells derived from the mutant mice exhibited premature senescence, spontaneously accumulated damaged chromosomes, and were sensitive to DNA crosslinking agents, but not to UV radiation. Genetic complementation studies revealed a crucial requirement for Slx4 to interact with the structure-specific endonuclease Ercc1 to promote crosslink repair.
In a Dutch boy, born of consanguineous parents, with Fanconi anemia of complementation group P (FANCP; 613951), Stoepker et al. (2011) identified a homozygous 1-bp deletion (286delA) in exon 1 of the SLX4 gene, resulting in a frameshift and premature termination at codon 126. Patient lymphoblasts showed spontaneous chromosomal breakage as well as chromosomal breakage in response to mitomycin C and camptothecin. Immunoassays of patient lymphoblasts did not detect a full-length protein, but there was a small amount of residual truncated protein identified with antibodies against the C terminus, which may have been derived from an alternative translation initiation site at codon 213 in the SLX4 gene. Transient expression in HEK293 cells of a truncated protein starting at met213 showed that it was able to interact with ERCC1 (126380), MUS81 (606591)-EME1 (610885), and SLX1. The cellular defects in this patient were believed to result from low levels of a truncated protein, resulting in a defect in the interaction of SLX4 with ERCC1. Transfection of a partial mouse Slx4 protein in patient cells partially restored chromosome breakage defects. The patient had growth retardation, mild dysmorphic features, hypoplastic right thumb, and pancytopenia.
In 3 German sibs with Fanconi anemia of complementation group P (FANCP; 613951), Stoepker et al. (2011) identified compound heterozygosity for 2 mutations in the SLX4 gene: a 1-bp deletion (1093delC), resulting in a frameshift and premature termination, and a splice site mutation in intron 5 (1163+3dupT; 613278.0003). The splice site mutation caused the skipping of exon 5 and resulted in an in-frame deletion disrupting the UBZ4 domain, which may be involved in targeting SLX4 to sites of DNA damage by binding to ubiquitinated proteins. Residual mutant protein in cell lysates was able to interact with ERCC4 (133520), ERCC1 (126380) and MUS81 (606591). Lymphoblasts from these individuals were sensitive to mitomycin C, but not to camptothecin, indicating that the mutant protein was partially functional. All 3 patients had pancytopenia.
For discussion of the splice site mutation in the SLX4 gene (1163+3dupT) that was found in compound heterozygous state in 3 sibs with Fanconi anemia of complementation group P (FANCP; 613951) by Stoepker et al. (2011), see 613278.0002.
In a 15-year-old girl from South India with Fanconi anemia of complementation group P (FANCP; 613951), Kim et al. (2011) identified a homozygous T-to-A transversion (1163+2T-A) in intron 5 of the SLX4 gene, resulting in the skipping of exon 5 and an in-frame deletion of the conserved cysteine and leucine of the first UBZ domain and the whole second UBZ domain. Immunoprecipitation studies of patient fibroblasts showed a slightly shorter protein product. Patient fibroblasts and lymphoblastoid cell lines showed increased genomic instability and increased sensitivity to mitomycin C; however, fibroblasts were not sensitive to camptothecin. Expression of wildtype SLX4 into patient fibroblasts rescued the phenotype. ERCC4, MUS81, and ERCC1 coimmunoprecipitated with the mutant protein in fibroblasts, but levels of the mutant protein were consistently lower in multiple experiments, leading to diminished immunoprecipitation of the interacting factors. The patient presented at age 9 years with isolated thrombocytopenia and had short stature and vitiligo.
In a 22-year-old man with Fanconi anemia of complementation group P (FANCP; 613951), Kim et al. (2011) identified compound heterozygosity for 2 mutations in the SLX4 gene: a 1-bp deletion (514delC) in exon 2, resulting in a frameshift and truncated protein, and a large genomic deletion extending from intron 9 to exon 12 (2013+225_3147del4890insCC; 613278.0006), predicted to result in a truncated protein. Immunoprecipitation studies failed to identify the full-length protein in patient fibroblasts. Patient fibroblasts showed increased genomic instability and increased sensitivity to mitomycin C and camptothecin; however, lymphoblastoid cell lines were not sensitive to mitomycin C, suggesting reversion. Expression of wildtype SLX4 into patient fibroblasts rescued the cellular phenotype. Overexpression of the 514delC-mutant protein in patient fibroblasts showed diminished, but present, interaction with ERCC4 (133520) and ERCC1 (126380), but not with MUS81 (606591). Immunoprecipitation of patient fibroblasts with an antibody recognizing the N terminus of SLX4 showed greatly diminished interaction with ERCC4, ERCC1, and MUS81. The patient was an American of European descent, and he had bilateral absent thumbs and right radial aplasia, pelvic kidney, undescended left testicle, malformed auricle, and short stature, as well as low platelets and anemia. He developed squamous cell carcinoma of the tongue at age 21 years and died at age 22.
For discussion of the splice site mutation in the SLX4 gene (2013+225_3147del4890insCC) that was found in compound heterozygous state in a patient with Fanconi anemia of complementation group P (FANCP; 613951) by Kim et al. (2011), see 613278.0005.
Crossan, G. P., van der Weyden, L., Rosado, I. V., Langevin, F., Gaillard, P.-H. L., McIntyre, R. E., Sanger Mouse Genetics Project, Gallagher, F., Kettunen, M. I., Lewis, D. Y., Brindle, K., Arends, M. J., Adams, D. J., Patel, K. J. Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia. Nature Genet. 43: 147-152, 2011. [PubMed: 21240276] [Full Text: https://doi.org/10.1038/ng.752]
Fekairi, S., Scaglione, S., Chahwan, C., Taylor, E. R., Tissier, A., Coulon, S., Dong, M.-Q., Ruse, C., Yates, J. R., III, Russell, P., Fuchs, R. P., McGowan, C. H., Gaillard, P.-H. L. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell 138: 78-89, 2009. [PubMed: 19596236] [Full Text: https://doi.org/10.1016/j.cell.2009.06.029]
Hartz, P. A. Personal Communication. Baltimore, Md. 2/19/2010.
Kim, Y., Lach, F. P., Desetty, R., Hanenberg, H., Auerbach, A. D., Smogorzewska, A. Mutations of the SLX4 gene in Fanconi anemia. Nature Genet. 43: 142-146, 2011. [PubMed: 21240275] [Full Text: https://doi.org/10.1038/ng.750]
Nagase, T., Kikuno, R., Ohara, O. Prediction of the coding sequences of unidentified human genes. XXII. The complete sequences of 50 new cDNA clones which code for large proteins. DNA Res. 8: 319-327, 2001. [PubMed: 11853319] [Full Text: https://doi.org/10.1093/dnares/8.6.319]
Nagase, T., Nakayama, M., Nakajima, D., Kikuno, R., Ohara, O. Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 8: 85-95, 2001. [PubMed: 11347906] [Full Text: https://doi.org/10.1093/dnares/8.2.85]
Stoepker, C., Hain, K., Schuster, B., Hilhorst-Hofstee, Y., Rooimans, M. A., Steltenpool, J., Oostra, A. B., Eirich, K., Korthof, E. T., Nieuwint, A. W. M., Jaspers, N. G. J., Bettecken, T., Joenje, H., Schindler, D., Rouse, J., de Winter, J. P. SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype. Nature Genet. 43: 138-141, 2011. [PubMed: 21240277] [Full Text: https://doi.org/10.1038/ng.751]
Svendsen, J. M., Smogorzewska, A., Sowa, M. E., O'Connell, B. C., Gygi, S. P., Elledge, S. J., Harper, J. W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell 138: 63-77, 2009. [PubMed: 19596235] [Full Text: https://doi.org/10.1016/j.cell.2009.06.030]
Wechsler, T., Newman, S., West, S. C. Aberrant chromosome morphology in human cells defective for Holliday junction resolution. Nature 471: 642-646, 2011. [PubMed: 21399624] [Full Text: https://doi.org/10.1038/nature09790]