Molecular Investigation of the Fanconi Anemia DNA repair pathway

© 2019 Winnie TanChemotherapeutic drugs often kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs) that inhibit DNA replication or transcription. Cells from patients with the genetic disorder Fanconi Anaemia (FA) are extremely sensitive to such DNA crosslinking agents. A critical step in the repair of ICLs is the biochemical modification of FANCI:FANCD2 protein by monoubiquitination. This monoubiquitination reaction is defective in 95% of FA patients, however the exact function of this process remains unclear. This thesis aims to better investigate how FANCI:FANCD2 monoubiquitination is regulated. To determine how monoubiquitinated proteins regulate the FA DNA repair pathway, several targets of monoubiquitination including FANCI:FANCD2, PCNA trimer and nucleosomes ubiquitinated by BRCA1:BARD1 were purified using Avi-ubiquitin. This thesis presents a minimal approach to purify challenging DNA repair proteins, which could potentially be applied to elucidate signalling mechanisms of other ubiquitination-mediated DNA repair pathways. Biochemical reconstitution of the FA pathway using recombinant human proteins revealed that FANCI:FANCD2 monoubiquitination is dependent on DNA and low ionic strength buffers. In addition, electrophoretic mobility shift assays revealed that monoubiquitination locks FANCI:FANCD2 on double-stranded DNA (dsDNA). Affinity pulldown of monoubiquitinated FANCI:FANCD2 confirmed that the complex remained bound to dsDNA, reflecting the monoubiquitination’s role in stabilising the FANCI:FANCD2 complex. The increased affinity of monoubiquitinated proteins on dsDNA suggests a conformational change on FANCI:FANCD2 after monoubiquitination. Using a newly developed Avi-tag-ubiquitin construct, the first successful purification of dually-monoubiquitinated FANCI:FANCD2 complex bound to DNA was reported. Strikingly, single-particle electron microscopy analysis revealed that monoubiquitinated FANCI:FANCD2 forms oligomers on dsDNA. This unexpected discovery hints that the function of monoubiquitination is to lock FANCI:FANCD2 oligomers on chromatin to initiate DNA repair. FANCI phosphorylation was previously reported to play a key role in stimulating FANCI:FANCD2 monoubiquitination. Mass spectrometry and western blot using phospho-specific antibodies revealed that ATR-mediated FANCI phosphorylation at specific serine-glutamate (SQ) clusters regulate both ubiquitination and deubiquitination of FANCI:FANCD2 complex. These results indicate a mechanistic view where FANCI phosphorylation is required for ubiquitination-mediated DNA repair which gives insights into the biology of FA pathway and potential chemosensitiser development. Collectively, the results presented in this thesis elucidate the role of monoubiquitination in locking FANCI:FANCD2 on DNA to form oligomers to initiate DNA repair. This work uncovers a new role of monoubiquitinated FANCI:FANCD2 during interstrand crosslink repair and provides new potential chemotherapeutic targets