Abstract
The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. 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.
Highlights
The E3 ligase FANCL monoubiquitinates FANCD2 in a critical step in the repair of DNA interstrand crosslinks
This study describes a hitherto unknown non-covalent interaction between FANCL and ubiquitin, with an affinity commonly observed in ubiquitin-protein interactions [36], that is required for efficient FANCD2 monoubiquitination
The interaction between the E2-like fold (ELF) domain of FANCL and ubiquitin is distinct from the surfaces in E2 proteins commonly used for non-covalent ubiquitin binding
Summary
The E3 ligase FANCL monoubiquitinates FANCD2 in a critical step in the repair of DNA interstrand crosslinks. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. 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. A key step in DNA ICL repair is the site-specific monoubiquitination of FANCD2 at Lys561 [10], which leads to the recruitment of downstream repair factors. This monoubiquitination event is carried out by FANCL, the RING E3 ligase subunit of the FA core complex [11, 12]. We show that the interaction is neither catalytic, nor required for complex formation, but is required for efficient FANCD2 monoubiquitination in cells
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