Abstract
DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair.
Highlights
Many chemotherapeutic drugs kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs)
Together using phospho-specific antibodies and mass spectrometry analysis (Supplementary Figure 1), we show that three FANCI serine residues are the substrates of ATR kinase, and are required for optimal monoubiquitination of FANCI and FANCD2
ATR kinase strongly influences the activation of the Fanconi Anemia DNA repair pathway (Andreassen et al, 2004)
Summary
Many chemotherapeutic drugs kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs). A critical step in the repair of replication forks stalled by ICLs is the biochemical modification of FANCD2 protein by mono-ubiquitination. Genetic deficiency in this pathway leads to Fanconi anemia (FA), characterized by hypersensitivity to DNA crosslinking agents, bone marrow failure, infertility, and cancer predisposition (Garaycoechea and Patel, 2014; Tsui and Crismani, 2019). FANCI itself is a target for monoubiquitination by the FA core complex (Smogorzewska et al, 2007), is a substrate of ATR kinase (Chen et al, 2015) and contains a USP1:UAF1 binding site necessary for deubiquitination of FANCD2 (Cohn et al, 2009). FANCD2 associates with FANCI as a heterodimer during ICL repair to signal DNA repair proteins that contain ubiquitin binding motifs, to promote DNA repair via homologous recombination or translesion synthesis (Smogorzewska et al, 2010; Klein Douwel et al, 2014)
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