The Fanconi Anemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks, and which is additionally activated in response to other forms of replication stress. A key step in the activation of the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double stranded DNA via promoting/stabilizing a large-scale conformational change in the complex, resulting in a secondary "Arm" ID2 interphase encircling DNA. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1/UAF deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilise a conformation in which the ID2 complex encircles DNA.
Ubiquitin-Specific Protease 1 (USP1), together with the cofactor UAF1, acts during DNA repair processes to specifically to remove mono-ubiquitin signals. The mono-ubiquitinated FANCI-FANCD2 heterodimer is one such substrate and is involved in the repair of DNA interstrand crosslinks via the Fanconi Anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin, and bound to mono-ubiquitinated FANCI-FANCD2 substrate. Crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1. A cryoEM reconstruction of USP1-UAF1 in complex mono-ubiquitinated FANCI-FANCD2, highlights a highly orchestrated deubiquitination process with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for their requirement despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition..
The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double‐stranded DNA via promoting a large‐scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary “Arm” ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1‐UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post‐translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.
Deubiquitination of FANCD2, FANCI, and PCNA by USP1 is essential for DNA repair signalling. Reconstitution of the system reveals that USP1-mediated specificity towards K561 of FANCD2 is directed by a unique sequence at USP1's N-terminus.
Ube2T is the E2 ubiquitin-conjugating
enzyme of the Fanconi anemia
DNA repair pathway and it is overexpressed in several cancers, representing
an attractive target for the development of inhibitors. Despite the
extensive efforts in targeting the ubiquitin system, very few E2 binders
have currently been discovered. Herein we report the identification
of a new allosteric pocket on Ube2T through a fragment screening using
biophysical methods. Several fragments binding to this site inhibit
ubiquitin conjugation in vitro.
DNA damage repair is implemented by proteins that are coordinated by specialised molecular signals. One such signal in the Fanconi Anemia (FA) DNA-interstrand crosslink repair pathway is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate E2 Ube2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyses ubiquitination by rewiring Ube2T's intra-residue network to influence the active site. Consequently, a basic triad unique to Ube2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of Ube2T can be engineered to enhance FANCL catalysed FANCD2-FANCI di-monoubiquitination without compromising site-specificity.
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