DNA interstrand-crosslinks (ICLs) are covalent links between complementary DNA strands that prevent their separation, interfering with fundamental cellular processes such as DNA replication and transcription. Consequently, these DNA lesions are highly toxic and the inability to repair them results in a severe genetic disease, Fanconi Anemia (FA), manifested by developmental impairment, bone marrow failure, and predisposition to various types of cancers. In healthy cells, a specialized cascade of DNA repair proteins comes together to establish the FA pathway that specifically recognizes and repairs DNA ICLs.

Ubiquitination of the FANCD2-FANCI (D2-I) complex by a multi-subunit ubiquitin E3 ligase, the FA core complex, is a key step of the FA pathway. D2-I ubiquitination initiates ICL repair by recruiting endonucleases to remove the DNA lesion, which is subsequently repaired via homologous recombination and trans-lesion synthesis. Due to its immense importance in the FA pathway, D2-I ubiquitination has been thoroughly studied using genetic, cellular, biochemical, and structural tools.

FANCI phosphorylation by the ATR DNA damage kinase stimulates D2-I ubiquitination in cells, which enables timely and coordinated FA pathway activation to ensure DNA repair fidelity. In this dissertation, I investigated the mechanism of this process by employing a triple-phosphomimetic FANCI. First, I confirmed the stimulatory effect of phosphomimetic FANCI on D2-I ubiquitination through in vitro ubiquitination assays using purified proteins. To elucidate the structural basis of this stimulation, I visualized different states of phosphomimetic D2-I using cryo-electron microscopy (cryo-EM). This revealed that phosphomimetic D2-I securely closes around double stranded DNA. Upon closure, target ubiquitination sites on D2-I become accessible, explaining how FANCI phosphorylation promotes D2-I ubiquitination. I further explored how phosphomimetic mutations affected biophysical properties and dynamics of the D2-I complex. Strikingly, while phosphomimetic mutations did not significantly alter DNA binding, in vitro ubiquitination assays suggested that phosphomimetic D2-I closes more readily in solution even in the absence of DNA. This is not due to increased binding of the phosphomimetic D2-I to the FA core complex. Instead, using quantitative crosslinking mass spectrometry, I discovered that phosphomimetic mutations locally alter the D2-I dimerization interface to prime the complex for closure. Thus, phosphomimetic mutations shift the conformational equilibrium of D2-I towards a closed state. Moreover, I observed that in the presence of DNA, phosphomimetic and wild-type D2-I exist in both open and closed states, however phosphomimetic D2-I has a higher preference for the closed conformation.

Overall, the work presented in this thesis provides mechanistic insight into how an interplay between DNA binding and FANCI phosphorylation by the ATR kinase alters the conformational dynamics of the D2-I complex, promoting its closure and ubiquitination to activate the FA pathway.