Homozygous mutations in the Fanconi Anemia DNA repair pathway pathway lead to Fanconi Anemia (FA), a devastating childhood genome instability disorder, typified by bone marrow failure and a high predisposition to cancers. The FA pathway is required for the repair of DNA interstrand crosslinks (ICLs), the hallmark of many cancers and FA. The FA pathway is regulated by ubiquitin, in a cycle of monoubiquitination and deubiquitination of FANCD2. Despite considerable advances in our understanding of the genetics of the pathway, there is strikingly little known on a mechanistic and chemical level concerning how the ubiquitin signal is assembled, recognised and disassembled. In particular, only one lysine on FANCD2 (K561) is targeted for monoubiquitination, despite having >60 lysines available on the surface. I will discuss recent findings that provide a structural and mechanistic rationale for the site-specific monoubiquitination of FANCD2.
Removal of the signal is also critical for completion of ICL repair, and requires the ubiquitin specific protease USP1, in complex with UAF1. Intriguingly, a second DNA repair pathway, the translesion synthesis pathway required for DNA damage tolerance, is also regulated by a cycle of monoubiquitination and deubiquitination of PCNA. Deubiquitination of PCNA requires the activity of USP1-UAF1 too.
Loss of USP1 activity gives rise to chromosomal instability. While many USPs hydrolyse ubiquitin-ubiquitin linkages, USP1 targets ubiquitin-substrate conjugates at specific sites. The molecular basis of USP1’s specificity for multiple substrates is poorly understood. I will describe the reconstitution of deubiquitination of purified monoubiquitinated FANCD2, FANCI and PCNA and describe our discovery that the molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. I will also show how this leads to specific signal removal, and discuss the implications for targeting multiple DNA repair pathways via ubiquitin signalling.