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The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

Miles JA, Frost MG, Carroll E, Rowe ML, Howard MJ, Sidhu A, Chaugule VK, Alpi AF, Walden H - J. Biol. Chem. (2015)

Bottom Line: The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown.We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro.However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.

View Article: PubMed Central - PubMed

Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.

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Ubiquitin binding is conserved in vertebrates.A, structure-based alignment of the ELF domain from various species of FANCL: Drosophila melanogaster (Dm), human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), and Danio rerio (Dr). Conserved residues are shaded red, conservative substitutions in orange, semi-conservative substitutions in yellow. Residues involved in ubiquitin-binding are boxed, and the Leu-81/Asn-72 residue is marked with an asterisk. Structural elements are included above the sequence. B, pull-down of Xenopus Tropicalis FANCL by ubiquitin shows that ubiquitin binding is conserved. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait and beads controls indicated.
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Figure 6: Ubiquitin binding is conserved in vertebrates.A, structure-based alignment of the ELF domain from various species of FANCL: Drosophila melanogaster (Dm), human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), and Danio rerio (Dr). Conserved residues are shaded red, conservative substitutions in orange, semi-conservative substitutions in yellow. Residues involved in ubiquitin-binding are boxed, and the Leu-81/Asn-72 residue is marked with an asterisk. Structural elements are included above the sequence. B, pull-down of Xenopus Tropicalis FANCL by ubiquitin shows that ubiquitin binding is conserved. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait and beads controls indicated.

Mentions: The residues in the ELF domain that are important for ubiquitin binding are not well conserved (Fig. 6A). However, ubiquitin-interacting proteins and motifs such as CUE domains, UBAs, UIMs, and MIUs often share very little sequence homology yet retain functional homology (33). We therefore wanted to determine whether the function of ubiquitin binding is conserved in vertebrate FANCL homologs. We and others had difficulties to make wild type full-length human FANCL, therefore we turned to the Xenopus system. Indeed. Xenopus tropicalis FANCL recapitulates ubiquitin binding (Fig. 6B). Mutation of Asn-72, corresponding to Leu-81 in the Drosophila protein, results in almost a complete loss of interaction with ubiquitin, indicating that the same region in FANCL is required for interaction with ubiquitin (Fig. 6B), and that the interaction is conserved between species.


The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

Miles JA, Frost MG, Carroll E, Rowe ML, Howard MJ, Sidhu A, Chaugule VK, Alpi AF, Walden H - J. Biol. Chem. (2015)

Ubiquitin binding is conserved in vertebrates.A, structure-based alignment of the ELF domain from various species of FANCL: Drosophila melanogaster (Dm), human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), and Danio rerio (Dr). Conserved residues are shaded red, conservative substitutions in orange, semi-conservative substitutions in yellow. Residues involved in ubiquitin-binding are boxed, and the Leu-81/Asn-72 residue is marked with an asterisk. Structural elements are included above the sequence. B, pull-down of Xenopus Tropicalis FANCL by ubiquitin shows that ubiquitin binding is conserved. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait and beads controls indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Ubiquitin binding is conserved in vertebrates.A, structure-based alignment of the ELF domain from various species of FANCL: Drosophila melanogaster (Dm), human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), and Danio rerio (Dr). Conserved residues are shaded red, conservative substitutions in orange, semi-conservative substitutions in yellow. Residues involved in ubiquitin-binding are boxed, and the Leu-81/Asn-72 residue is marked with an asterisk. Structural elements are included above the sequence. B, pull-down of Xenopus Tropicalis FANCL by ubiquitin shows that ubiquitin binding is conserved. Each experiment is probed with anti-His-ubiquitin and anti-FANCL antibodies, with the input, bait and beads controls indicated.
Mentions: The residues in the ELF domain that are important for ubiquitin binding are not well conserved (Fig. 6A). However, ubiquitin-interacting proteins and motifs such as CUE domains, UBAs, UIMs, and MIUs often share very little sequence homology yet retain functional homology (33). We therefore wanted to determine whether the function of ubiquitin binding is conserved in vertebrate FANCL homologs. We and others had difficulties to make wild type full-length human FANCL, therefore we turned to the Xenopus system. Indeed. Xenopus tropicalis FANCL recapitulates ubiquitin binding (Fig. 6B). Mutation of Asn-72, corresponding to Leu-81 in the Drosophila protein, results in almost a complete loss of interaction with ubiquitin, indicating that the same region in FANCL is required for interaction with ubiquitin (Fig. 6B), and that the interaction is conserved between species.

Bottom Line: The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown.We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro.However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.

View Article: PubMed Central - PubMed

Affiliation: From the Protein Structure and Function Laboratory, Lincoln's Inn Fields Laboratories of the London Research Institute, Cancer Research, United Kingdom, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.

Show MeSH
Related in: MedlinePlus