Although FANCL is active in isolation, its catalytic function is greatly stimulated by the interaction with FANCB and FAAP100 ( Huang et al., 2014, Rajendra et al., 2014). FANCL is an E3 ligase containing a RING domain that is essential for the interaction with the E2 enzyme, UBE2T/FANCT, and for the mono-ubiquitination of the FANCI and FANCD2 molecular targets ( Cole et al., 2010, Hodson et al., 2011, Hodson et al., 2014, Machida et al., 2006).

While the AG20 and CEF play loosely defined ancillary roles ( Wang and Smogorzewska, 2015), the BL100 complex is a three-member ubiquitin ligase, with FANCL as the minimal unit required for ubiquitination ( Huang et al., 2014, Meetei et al., 2003, Rajendra et al., 2014). The core complex is composed of three distinct protein assemblies with non-redundant functions: FANCB-FANCL-FAAP100 (hereinafter called BL100), FANCC-FANCE-FANCF (CEF), and FANCA-FANCG-FAAP20 (AG20) ( Huang et al., 2014, Rajendra et al., 2014). A key player in the recognition of ICLs is the FA anchor complex (consisting of the FANCM DNA translocase, FAAP24, and two histone-like factors named MHF1-2), which, in turn, recruits the FA core complex to the DNA damage site ( Ciccia et al., 2007, Coulthard et al., 2013, Kim et al., 2008, Yan et al., 2010). Mutations in the FA genes cause a rare genetic disorder characterized by hypersensitivity to crosslinking agents, chromosomal instability, and cancer predisposition ( Deans and West, 2011).ĭespite the enormous effort of several research groups, a mechanistic understanding of the activation of the FA ICL repair pathway is only starting to emerge ( Walden and Deans, 2014).

Activation of this pathway depends on the specific mono-ubiquitination of the FANCI and FANCD2 proteins by the FA core complex ( Ceccaldi et al., 2016, Garcia-Higuera et al., 2001, Sims et al., 2007, Smogorzewska et al., 2007, Timmers et al., 2001). The Fanconi anemia (FA)/BRCA pathway is the major interstrand crosslink repair process in higher eukaryotes ( Knipscheer et al., 2009, Kottemann and Smogorzewska, 2013). Interstrand crosslinks (ICLs) are toxic DNA lesions that prevent the separation of the two filaments in the DNA double helix, impairing DNA replication and transcription. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. How FANCI mono-ubiquitination is achieved remains unclear. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism.

FANCI and FANCD2 form a stable complex however, the molecular basis of their ubiquitination is ill defined. Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex.

Fa sds drop box