Regulation of the Fanconi Anemia pathway in human cells

Thesis

The Fanconi Anemia (FA) pathway is an essential DNA repair pathway responsible for resolving genotoxic DNA interstrand crosslinks (ICLs). Activation of the FA pathway occurs when a replication fork stalls at an ICL lesion, leading to the recruitment of a heterodimeric FANCD2–FANCI complex to the damage site. This complex promotes repair through coordinated strand excision, translesion DNA synthesis, and homologous recombination. Numerous DNA damage response and repair proteins participate in this process, and both activation and deactivation of the FA pathway are subject to stringent regulatory control. However, the molecular mechanisms underlying this process remain incompletely understood. In this thesis, a live-cell single-molecule tracking approach was developed and applied to investigate the dynamic behavior of FANCD2 during ICL repair. By tracking individual FANCD2 molecules in cells with or without ICL damage, I show that phosphorylation mediated by the ATR kinase and dephosphorylation by the PP2A phosphatase are both required during FA pathway activation. Together, these opposing regulatory activities facilitate a transition of FANCD2 molecules from a mobile to a more immobile state, consistent with its stable loading onto ICL-damaged chromatin. This work establishes a quantitative framework for studying the FA pathway at the single-molecule level in live human cells and demonstrates the broader applicability of this approach for dissecting complex DNA repair processes in vivo.

In addition, a protein previously characterized for its role in mitotic and cell-cycle regulation is identified here as a novel factor required for activation of the FA pathway, without detectable effects on overall cell-cycle progression. Recruitment of this protein to ICL damage sites promotes FANCD2 foci formation and subsequent monoubiquitination. Although the precise molecular mechanism remains to be elucidated, these findings provide functional evidence supporting a role for this candidate protein in ICL repair.

Taken together, these findings support a model in which FA pathway activation is governed by multilayered and dynamic regulatory mechanisms rather than static protein abundance, providing new mechanistic insight with potential relevance for future biological and clinical studies.

Authors: Bai, F

• DOI: 10.5287/ora-garerdyae
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: DNA interstrand crosslink repair; genome stability; FANCD2; Fanconi anemia pathway; single-molecule tracking; FANCD2/FANCI complex
• Subjects: Biological science; Molecular biology