Abstract

Fanconi Anemia (FA) is a rare human genetic disease that is characterized by birth defects, progressive bone marrow failure and predisposition to cancer, such as acute myeloid leukemia and squamous cell carcinomas. At the cellular level, FA is characterized by chromosomal instability and hypersensitivity to agents that cause DNA interstrand cross-links (ICL), such as mitomycin C (MMC) and diepoxybutane. Up to date thirteen FA genes corresponding to thirteen FA complementation groups have been identified, and the protein products of them constitute a novel pathway that is involved in repairing DNA ICLs. Among the thirteen FA proteins, eight of them (FANCA, B, C, E, F, G, L and M) form a constitutive nuclear protein complex with ubiquitin ligase activity. Upon DNA damage this FA complex monoubiquitinates the FA proteins I and D2, which form an ID complex in the nucleus. The monoubiquitinated ID complex targets to DNA damage foci and cooperates with DNA repair proteins such as BRCA1, BRCA2, RAD51 and others. To further investigate the biological functions of the FA proteins in ICL repair, I purified recombinant proteins of the C-terminal domain (CTD) of FANCF; the N-terminal domain (NTD) of FANCA in complex with FANCG; and the full-length FANCD2 and FANCI proteins. I was able to crystallize FANCF-CTD, FANCA(NTD)/FANCG and FANCI and solved the 3-dimensional structures of the FANCF-CTD and FANCI proteins. Both the structures of FANCI and FANCF-CTD consist of novel FANC two-helix repeats. Analysis of crystal packing interactions, taken together with amino acid sequence conservation suggests a possible binding interface between FANCI and FANCD2. The mapping of conserved residues onto the surface of the FANCI structure revealed a conserved ubiquitination site surface patch, a finding which suggests the requirement of amino acids other than K522 for monoubiquitination of the protein. FANCI contains a positively charged surface groove that predicts that FANCI may bind to double-stranded DNA (dsDNA). DNA binding assays confirmed that both FANCI and the ID complex possess dsDNA binding activity. The ID complex binds to dsDNA with minimal length requirement of about 15 base pairs with no significant single-stranded DNA binding activity. Our studies give structural insights into the FA proteins and suggest a direct role for the ID complex in DNA binding, which is consistent with its role in DNA damage repair.