The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2

摘要

class="head no_bottom_margin" id="sec1title">IntroductionInterstrand crosslinks (ICLs) are toxic DNA lesions that prevent the separation of the two filaments in the DNA double helix, impairing DNA replication and transcription. The Fanconi anemia (FA)/BRCA pathway is the major interstrand crosslink repair process in higher eukaryotes (, ). Activation of this pathway depends on the specific mono-ubiquitination of the FANCI and FANCD2 proteins by the FA core complex (, , , , ). Mutations in the FA genes cause a rare genetic disorder characterized by hypersensitivity to crosslinking agents, chromosomal instability, and cancer predisposition ().Despite the enormous effort of several research groups, a mechanistic understanding of the activation of the FA ICL repair pathway is only starting to emerge (). 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 (, , , ). 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) (, ). While the AG20 and CEF play loosely defined ancillary roles (href="#bib50" rid="bib50" class=" bibr popnode">Wang and Smogorzewska, 2015), the BL100 complex is a three-member ubiquitin ligase, with FANCL as the minimal unit required for ubiquitination (href="#bib19" rid="bib19" class=" bibr popnode">Huang et al., 2014, href="#bib32" rid="bib32" class=" bibr popnode">Meetei et al., 2003, href="#bib39" rid="bib39" class=" bibr popnode">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 (href="#bib9" rid="bib9" class=" bibr popnode">Cole et al., 2010, href="#bib17" rid="bib17" class=" bibr popnode">Hodson et al., 2011, href="#bib18" rid="bib18" class=" bibr popnode">Hodson et al., 2014, href="#bib31" rid="bib31" class=" bibr popnode">Machida et al., 2006). Although FANCL is active in isolation, its catalytic function is greatly stimulated by the interaction with FANCB and FAAP100 (href="#bib19" rid="bib19" class=" bibr popnode">Huang et al., 2014, href="#bib39" rid="bib39" class=" bibr popnode">Rajendra et al., 2014). Whether binding of these two factors causes an activating conformational switch in the FANCL catalytic center, or whether FANCB and FAAP100 play an architectural role important for mono-ubiquitination, is unknown.Multiple other architectural aspects of the mono-ubiquitination of FANCI and FANCD2 likewise remain poorly understood. First, crystallographic studies on mouse proteins indicate that the FANCI-FANCD2 (ID2) hetero-dimer forms a pseudo-2-fold symmetric arrangement. However, the mono-ubiquitination sites map at the dimerization interface within the ID2 complex (href="#bib21" rid="bib21" class=" bibr popnode">Joo et al., 2011), suggesting that a reconfiguration of this interface is needed at some point during the mono-ubiquitination reaction to allow for access of the E3 ligase assembly. Second, whether a stable ID2 complex is recruited to the DNA damage site prior to mono-ubiquitination is unclear, although recent cryoelectron microscopy (cryo-EM) studies on the unmodified human proteins support this notion (href="#bib28" rid="bib28" class=" bibr popnode">Liang et al., 2016). Remarkably the symmetric hetero-dimer captured by crystallography cannot be recognized in the electron microscopy (EM) structure, suggesting that the ID2 complex might visit different conformational states throughout the ubiquitination reaction. Finally, although both members of the ID2 dimer need to be mono-ubiquitinated for activation of the ICL repair pathway (href="#bib44" rid="bib44" class=" bibr popnode">Sims et al., 2007, href="#bib45" rid="bib45" class=" bibr popnode">Smogorzewska et al., 2007, href="#bib47" rid="bib47" class=" bibr popnode">Timmers et al., 2001), so far, biochemical reconstitution has mainly focused on the minimal ability of the BL100 E3 ligase to mono-ubiquitinate FANCD2, with little to no FANCI mono-ubiquitination observed (href="#bib39" rid="bib39" class=" bibr popnode">Rajendra et al.,2014). How equivalent mono-ubiquitination of both FANCI and FANCD2 is achieved and whether any of the ancillary proteins in the FA core complex are involved in this process remain unresolved questions.To address these issues, we have used EM, combined with crosslinking and mass spectrometry (XL/MS), to analyze the architecture of the BL100 complex and its substrate ID2, either in isolation or with other components of the FA core complex. We found that BL100 forms a symmetric dimer of trimers, inviting a molecular model for the equivalent ubiquitination of the dimeric ID2 assembly. The CEF component of the FA core complex binds to the two extremities in the BL100 dimer, probably acting as a molecular matchmaker that mediates the BL100 and ID2 interaction. We also uncovered an unexpected structural function for CEF, which stabilizes the dimerization interface of the ID2 complex. We postulate that this function might have a direct role in the efficient mono-ubiquitination of the ID2 hetero-dimeric substrate. Our findings provide key insights into the structural basis for the mono-ubiquitination of the ID2 complex, the defining step in the activation of the FA/BRCA ICL repair pathway.