Mono-ubiquitination by the FA core complex clamps FANCI:FANCD2 on DNA as a filamentous array Winnie Tan1, Sylvie van Twest1, Vincent Murphy2, Andrew Deans1,2 1Genome Stability Unit, St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, 3065 Australia; 2Department of Medicine (St. Vincent’s Health), The University of Melbourne, Australia The Fanconi Anemia (FA) pathway is required for efficient repair of interstrand crosslink DNA damage. In response to DNA damage, the FA core complex containing the RING-E3 ligase activates the mono-ubiquitination of FANCI:FANCD2 to stabilise replication forks. The central molecular defect of FA is absence of the mono-ubiquitinated form of FANCD2 and its partner protein FANCI. The biochemical function of FANCI:FANCD2 mono-ubiquitination is unclear, although it is required for protection of nascent DNA from degradation at stalled replication forks. It has been proposed that at stalled replication forks, mono-ubiquitinated FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. We show that mono-ubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilises FANCI:FANCD2 heterodimers on dsDNA. This clamping of FANCI:FANCD2 complex on DNA requires mono-ubiquitination of only the FANCD2 subunit. We further show that purified mono-ubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Mono-ubiquitination of both FANCI and FANCD2 are required to form this higher order structure, suggesting it may have a structural role in stabilising stalled replication forks.