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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 not required for E2 recognition.A, pull-down analysis of the interaction between wild type and L81R Drosophila FANCL and human Ube2T or Ube2T-Ub. Both FANCL species bound Ube2T and Ube2T-Ub to the same extent. B, Western blot analysis of Ube2T autoubiquitination in the absence and presence of Drosophila FANCL WT, L81R, and ΔELF species. All variations of E3 were able to successfully stimulate discharge of ubiquitin from Ube2T onto itself. C, in-gel fluorescence analysis of in vitro FANCD2 monoubiquitination (left). Ubiquitin is fluorescently labeled, with no ATP and no E3 controls, showing the modification of FANCD2. The right panel shown 5 independent replicates, with a Coomassie-stained loading control, and quantification of the level of FANCD2 ubiquitination (bottom).
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Figure 7: Ubiquitin binding is not required for E2 recognition.A, pull-down analysis of the interaction between wild type and L81R Drosophila FANCL and human Ube2T or Ube2T-Ub. Both FANCL species bound Ube2T and Ube2T-Ub to the same extent. B, Western blot analysis of Ube2T autoubiquitination in the absence and presence of Drosophila FANCL WT, L81R, and ΔELF species. All variations of E3 were able to successfully stimulate discharge of ubiquitin from Ube2T onto itself. C, in-gel fluorescence analysis of in vitro FANCD2 monoubiquitination (left). Ubiquitin is fluorescently labeled, with no ATP and no E3 controls, showing the modification of FANCD2. The right panel shown 5 independent replicates, with a Coomassie-stained loading control, and quantification of the level of FANCD2 ubiquitination (bottom).

Mentions: Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (34, 35). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T∼Ub). To test this hypothesis, we generated a stable Ube2T∼Ub ester and assayed FANCL binding via pull-down. We observed no difference in the levels of FANCL binding in a comparison between ubiquitin-charged Ube2T and uncharged Ube2T (Fig. 7A). Furthermore, there was no difference in Ube2T∼Ub binding when the ubiquitin binding surface of ELF was mutated. These data suggest that ubiquitin binding does not enhance FANCL's interaction with E2.


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 not required for E2 recognition.A, pull-down analysis of the interaction between wild type and L81R Drosophila FANCL and human Ube2T or Ube2T-Ub. Both FANCL species bound Ube2T and Ube2T-Ub to the same extent. B, Western blot analysis of Ube2T autoubiquitination in the absence and presence of Drosophila FANCL WT, L81R, and ΔELF species. All variations of E3 were able to successfully stimulate discharge of ubiquitin from Ube2T onto itself. C, in-gel fluorescence analysis of in vitro FANCD2 monoubiquitination (left). Ubiquitin is fluorescently labeled, with no ATP and no E3 controls, showing the modification of FANCD2. The right panel shown 5 independent replicates, with a Coomassie-stained loading control, and quantification of the level of FANCD2 ubiquitination (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Ubiquitin binding is not required for E2 recognition.A, pull-down analysis of the interaction between wild type and L81R Drosophila FANCL and human Ube2T or Ube2T-Ub. Both FANCL species bound Ube2T and Ube2T-Ub to the same extent. B, Western blot analysis of Ube2T autoubiquitination in the absence and presence of Drosophila FANCL WT, L81R, and ΔELF species. All variations of E3 were able to successfully stimulate discharge of ubiquitin from Ube2T onto itself. C, in-gel fluorescence analysis of in vitro FANCD2 monoubiquitination (left). Ubiquitin is fluorescently labeled, with no ATP and no E3 controls, showing the modification of FANCD2. The right panel shown 5 independent replicates, with a Coomassie-stained loading control, and quantification of the level of FANCD2 ubiquitination (bottom).
Mentions: Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (34, 35). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T∼Ub). To test this hypothesis, we generated a stable Ube2T∼Ub ester and assayed FANCL binding via pull-down. We observed no difference in the levels of FANCL binding in a comparison between ubiquitin-charged Ube2T and uncharged Ube2T (Fig. 7A). Furthermore, there was no difference in Ube2T∼Ub binding when the ubiquitin binding surface of ELF was mutated. These data suggest that ubiquitin binding does not enhance FANCL's interaction with E2.

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