Abstract
SummaryFanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
Highlights
Fanconi anaemia (FA) is a rare, recessive disorder with a striking phenotype consisting of developmental defects, bone marrow failure, and cancer predisposition (Crossan and Patel, 2012)
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers
We report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity
Summary
Fanconi anaemia (FA) is a rare, recessive disorder with a striking phenotype consisting of developmental defects, bone marrow failure, and cancer predisposition (Crossan and Patel, 2012). Five FA-associated proteins (FAAPs) play roles in the FA DNA repair pathway. After DNA damage, this multisubunit E3 ligase carries out the specific monoubiquitination of FANCI and FANCD2, a critical event that initiates DNA repair (Garcia-Higuera et al, 2001; Knipscheer et al, 2009; Smogorzewska et al, 2007). Ubiquitinated FANCD2 is a platform for the recruitment of additional proteins that coordinate DNA repair. These include the FAN1 nuclease and the scaffold protein FANCP (SLX4), which recruits the XPF-ERCC1, SLX1, and MUS81-EME1 nucleases (Cybulski and Howlett, 2011; Huang and D’Andrea, 2010)
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