Fanconi anaemia (FA) is a genetic disorder characterised by bone marrow failure, cancer predisposition and extreme sensitivity to DNA interstrand crosslinking agents. FA can be caused by deficiency in any one of twenty-two proteins that all act in a common “FA pathway” of DNA repair. At the centre of this pathway is the mono-ubiquitination of FANCI:FANCD2 heterodimer by a “FA core complex” of nine proteins, at the sites of damage. Despite being used for 20 years as a marker for the activity of the FA pathway, and diagnostic for FA, the biochemical function of FANCI:FANCD2 mono-ubiquitination was unclear. At a cellular level 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.
Using a baculovirus co-expression system, we have biochemically reconstituted the entire FA pathway using recombinant proteins (1). 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 (2). 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. Our findings demonstrate that filamentous array formation on DNA is a critical function of the FA pathway in maintaining genome stability.