The Fanconi anemia (FA) pathway is required for efficient repair of interstrand crosslink (ICL) DNA damage. Central to the FA pathway is monoubiquitination of FANCI:FANCD2 by the FA core complex, a large E3 ligase. Currently it remains unclear how monoubiquitination of FANCI:FANCD2 activates a downstream ICL repair pathway.
We have developed a biochemical system that reconstitutes the FA pathway in vitro with >10 human proteins. This allowed us to study monoubiquitination by the FA core complex and its effect on FANCI:FANCD2 function. We show that monoubiquitination of FANCI:FANCD2 is robustly stimulated by different DNA structures. When the affinity of monoubiquitinated and unmodified (apo)-FANCI:FANCD2 was compared using electromobility shift assay (EMSA), we observed monoubiquitinated FANCI:FANCD2 complex stabilized on DNA. We also confirmed that the gel shifted band contains both FANCI and FANCD2 proteins, confirming that the proteins act as a dimeric complex during DNA interactions.
Addition of competitor DNA after FANCI:FANCD2 was monoubiquitinated prevented it from being displaced, but addition before monoubiquitination caused a concentration-dependent inhibition of EMSA shift. This result can be explained by a model where apo-FANCI:FANCD2 moves freely between the unlabelled and labelled DNA molecules, but after monoubiquitination, it is immobilized on the labelled DNA. Reversal of the ubiquitination reaction by the USP1:UAF1 deubiquitinase unlocks FANCI:FANCD2 from the DNA. It is then free to move to new substrates. This result suggests that USP1:UAF1 plays a critical role in the transport of FANCI:FANCD2 on damaged DNA, and the prevention of abnormal activation of the FA pathway.
Together, our results demonstrate that monoubiquitination of FANCI:FANCD2 complex “locks” it on DNA. We are currently exploring how the locking mechanism controls DNA repair, either through direct stabilization of DNA repair intermediates and/or the recruitment of additional factors.