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Mutations of the SLX4 gene in Fanconi anemia.

Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A - Nat. Genet. (2011)

Bottom Line: Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors.Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents.Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

ABSTRACT
Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

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Interaction of mutant forms of SLX4 with its partners and with ubiquitin. A. Analysis of SLX4 interacting partners in SLX4 mutant cells. Cell extracts of primary fibroblasts (BJ, RA3083, and RA3331) were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. B. Analysis of SLX4 interacting partners in RA3331 cells. Cell extracts of RA3331 E6E7 cells expressing HA tagged control vector (CONTROL), WT SLX4, or the p.L672VfsX119 SLX4 were subjected to immunoprecipitation using HA antibody or HA antibody in the presence of 30 µg of HA peptide. Interacting proteins were identified by immunoblotting with the indicated antibodies. C. Interaction of the UBZ domains with ubiquitin. GST-purified GST control, WT UBZ domains (SLX4aa 251-402) and UBZ domains with four cysteines mutated to alanines (SLX4aa251-402_C296A_C299A_C336A_C339A) were incubated with the indicated forms of ubiquitin, purified by binding to GST beads, separated on a PAGE gel and immunobloted with anti-ubiquitin antibody. The bottom panel shows Ponceau staining of the GST proteins.
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Figure 4: Interaction of mutant forms of SLX4 with its partners and with ubiquitin. A. Analysis of SLX4 interacting partners in SLX4 mutant cells. Cell extracts of primary fibroblasts (BJ, RA3083, and RA3331) were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. B. Analysis of SLX4 interacting partners in RA3331 cells. Cell extracts of RA3331 E6E7 cells expressing HA tagged control vector (CONTROL), WT SLX4, or the p.L672VfsX119 SLX4 were subjected to immunoprecipitation using HA antibody or HA antibody in the presence of 30 µg of HA peptide. Interacting proteins were identified by immunoblotting with the indicated antibodies. C. Interaction of the UBZ domains with ubiquitin. GST-purified GST control, WT UBZ domains (SLX4aa 251-402) and UBZ domains with four cysteines mutated to alanines (SLX4aa251-402_C296A_C299A_C336A_C339A) were incubated with the indicated forms of ubiquitin, purified by binding to GST beads, separated on a PAGE gel and immunobloted with anti-ubiquitin antibody. The bottom panel shows Ponceau staining of the GST proteins.

Mentions: SLX4 interacts with multiple factors, two of which, ERCC4/XPF and MUS81 have been previously implicated in crosslink repair8. We therefore tested whether the mutant SLX4 proteins from both patients still interacted with the ERCC4/XPF and MUS81 complexes. We found that ERCC4/XPF, MUS81, and ERCC1 co-immunoprecipitate with endogenous mutant SLX4 (p.R317_F387del) from RA3083 fibroblasts (Figure 4A, lane 5 and Figure S5A, lane 4), although the levels of the mutant SLX4 protein were consistently lower in multiple experiments leading to diminished immunoprecipitation of the interacting factors. The second patient’s SLX4 p.L672VfsX119 overexpressed in RA3331 fibroblasts showed diminished but present interaction with ERCC4/XPF and ERCC1, but not with MUS81 (Figure 4B, lane 3). This was consistent with the previous findings that MUS81 interacts with aa 684–1834 fragment of the SLX4 protein7 which are deleted in the p.L672VfsX119 mutant protein. Immunoprecipitation with an antibody recognizing the N terminus of SLX4 from RA3331 cells showed greatly diminished interaction with ERCC4/XPF, ERCC1, and MUS81 (Figure S5B, lane 6).


Mutations of the SLX4 gene in Fanconi anemia.

Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A - Nat. Genet. (2011)

Interaction of mutant forms of SLX4 with its partners and with ubiquitin. A. Analysis of SLX4 interacting partners in SLX4 mutant cells. Cell extracts of primary fibroblasts (BJ, RA3083, and RA3331) were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. B. Analysis of SLX4 interacting partners in RA3331 cells. Cell extracts of RA3331 E6E7 cells expressing HA tagged control vector (CONTROL), WT SLX4, or the p.L672VfsX119 SLX4 were subjected to immunoprecipitation using HA antibody or HA antibody in the presence of 30 µg of HA peptide. Interacting proteins were identified by immunoblotting with the indicated antibodies. C. Interaction of the UBZ domains with ubiquitin. GST-purified GST control, WT UBZ domains (SLX4aa 251-402) and UBZ domains with four cysteines mutated to alanines (SLX4aa251-402_C296A_C299A_C336A_C339A) were incubated with the indicated forms of ubiquitin, purified by binding to GST beads, separated on a PAGE gel and immunobloted with anti-ubiquitin antibody. The bottom panel shows Ponceau staining of the GST proteins.
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Related In: Results  -  Collection

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Figure 4: Interaction of mutant forms of SLX4 with its partners and with ubiquitin. A. Analysis of SLX4 interacting partners in SLX4 mutant cells. Cell extracts of primary fibroblasts (BJ, RA3083, and RA3331) were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. B. Analysis of SLX4 interacting partners in RA3331 cells. Cell extracts of RA3331 E6E7 cells expressing HA tagged control vector (CONTROL), WT SLX4, or the p.L672VfsX119 SLX4 were subjected to immunoprecipitation using HA antibody or HA antibody in the presence of 30 µg of HA peptide. Interacting proteins were identified by immunoblotting with the indicated antibodies. C. Interaction of the UBZ domains with ubiquitin. GST-purified GST control, WT UBZ domains (SLX4aa 251-402) and UBZ domains with four cysteines mutated to alanines (SLX4aa251-402_C296A_C299A_C336A_C339A) were incubated with the indicated forms of ubiquitin, purified by binding to GST beads, separated on a PAGE gel and immunobloted with anti-ubiquitin antibody. The bottom panel shows Ponceau staining of the GST proteins.
Mentions: SLX4 interacts with multiple factors, two of which, ERCC4/XPF and MUS81 have been previously implicated in crosslink repair8. We therefore tested whether the mutant SLX4 proteins from both patients still interacted with the ERCC4/XPF and MUS81 complexes. We found that ERCC4/XPF, MUS81, and ERCC1 co-immunoprecipitate with endogenous mutant SLX4 (p.R317_F387del) from RA3083 fibroblasts (Figure 4A, lane 5 and Figure S5A, lane 4), although the levels of the mutant SLX4 protein were consistently lower in multiple experiments leading to diminished immunoprecipitation of the interacting factors. The second patient’s SLX4 p.L672VfsX119 overexpressed in RA3331 fibroblasts showed diminished but present interaction with ERCC4/XPF and ERCC1, but not with MUS81 (Figure 4B, lane 3). This was consistent with the previous findings that MUS81 interacts with aa 684–1834 fragment of the SLX4 protein7 which are deleted in the p.L672VfsX119 mutant protein. Immunoprecipitation with an antibody recognizing the N terminus of SLX4 from RA3331 cells showed greatly diminished interaction with ERCC4/XPF, ERCC1, and MUS81 (Figure S5B, lane 6).

Bottom Line: Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors.Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents.Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

ABSTRACT
Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

Show MeSH
Related in: MedlinePlus