Fanconi Anemia (FA) is a multigenic autosomal recessive syndrome characterized by multiple congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. Increasing evidence indicates the FA proteins cooperate in a novel DNA damage response pathway. A key step in this pathway is the monoubiquitination of FANCD2, resulting in its redistribution into nuclear foci. FA cells are exquisitely sensitive to interstrand cross-linkers, such as Mitomycin C (MMC), but relatively resistant to ionizing radiation (IR), although both mutagens generate double-strand breaks (DSBs) and activate the FA pathway. This suggests that, despite its general activation in response to DSBs; the FA pathway is specifically required for the damage response to MMC induced lesions. Even with major advances in our molecular understanding of the FA pathway, little is known about its function. Several FA proteins (A/C/E/F/G/L) are components of a multi-subunit complex required for the monoubiquitination of another FA protein, FANCD2 in response to DNA damage. Upon monoubiquitination, the activated FANCD2 (FANCD2-Ub) redistributes to nuclear foci where it colocalizes with several proteins implicated in homology-dependent repair (HDR). Virtually nothing is known about the purpose of the FA pathway following formation of FANCD2 foci. It is my hypothesis that, having no obvious functional domains of its own, FANCD2 facilitates repair of DNA damage by influencing the function of known repair pathways, such as the HDR pathway. To resolve the function of the FA pathway, it is essential to discern the identities of other gene products and, potentially, other repair pathways that may interact with FANCD2 and determine how FANCD2 influences these pathways in the repair of MMC induced interstrand cross-linked lesions. Taking advantage of the existing expertise in the D'Andrea lab in cell biology, and our comprehensive FA cell repository and applying techniques learned in collaboration with leaders in the field of chromatin biology, I propose a multifaceted approach to analyze the role of the FANCD2 protein in the repair of cross-linked DNA. Specifically, I propose to (i) Characterize the factors that bind to FANCD2 following monoubiquitination and foci formation; (ii) Identify proteins that bind differentially to FANCD2 following activation of the FA pathway in wild-type and select FA patient cell lines; (iii) Develop in vitro assays, to evaluate how FANCD2 interacts with DNA, and whether it can influence HDR repair assays involving BRCA2-mediated RAD51 filament formation and strand exchange.
|Lizarraga, Sofia B; Margossian, Steven P; Harris, Marian H et al. (2010) Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors. Development 137:1907-17|