Browsing by Subject "FANCD2"

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    Novel roles for the Fanconi Anemia pathway protein FANCD2 in the recovery of stalled replication forks
    (2017-06) Raghunandan, Maya
    Fanconi Anemia (FA) is an inherited cancer predisposition syndrome that is characterized by a cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these DNA lesions, the 21 known FA proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex activates two central FA pathway members, FANCD2 and FANCI, via monoubiquitination. Both activated proteins then bind the ICL and recruit downstream FA proteins that repair the ICLs. Importantly, we previously found that FANCD2 has an additional independent role during the cellular replication stress response: it promotes the homologous recombination (HR) dependent restart of hydroxyurea (HU) stalled replication forks in concert with other HR DNA repair proteins such as the BLM helicase. In this work, we show that FANCD2 promotes replication fork restart in concert with downstream FA pathway proteins but independently of the upstream FA core complex and thus, independently of FANCD2 monoubiquitination. To further our understanding of how FANCD2 promotes replication fork recovery, we performed a search for S-phase specific FANCD2 interactors and we identified a novel FANCD2 interacting protein, Alpha Thalassemia Retardation X-linked factor (ATRX). ATRX is a subunit of the ATRX/DAXX histone H3 chaperone complex that plays several key roles in regulating chromatin structure and was recently identified as a replication fork recovery factor. Our new findings demonstrate that ATRX forms a constitutive complex with FANCD2 and promotes FANCD2 protein stability. Moreover, while ATRX is dispensable for DNA ICL repair, it works in concert with FANCD2 to promote HU resistance and the restart of HU-stalled replication forks. Remarkably, the HR-dependent replication fork restart requires the histone H3 chaperone activity of both the ATRX/DAXX complex and FANCD2 indicating that histone exchange at stalled replication forks is a crucial step in fork restart. Altogether, our results support a novel non-linear FA pathway model where individual protein members fulfill distinct cellular roles to support genomic stability. We propose that FANCD2- and possibly other FA pathway proteins- is involved in the deposition of histone H3 variants in the vicinity of HU- stalled replication forks to mediate fork recovery.
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    The Role of Fanconi Anemia Proteins in DNA Repair, Replication Stress and Genome Stability
    (2017-10) Thompson, Elizabeth
    Fanconi anemia (FA) is a genetic chromosomal instability disorder characterized by progressive bone marrow failure and a strong predisposition to cancer. The FA proteins work together in a cellular pathway for the repair of DNA interstrand crosslinks (ICLs). Currently 22 different FA genes are implicated in this disease and contribute to the heterogeneity in symptoms and severity. Using gene targeting techniques, we successfully created a set of isogenic knockout cell lines to represents all 3 groups of proteins within the FA pathway to characterize protein function and identify differences that help explain FA disease heterogeneity. In Chapter 2 we investigate the FA group 2 proteins, FANCI and FANCD2 that that form a heterodimer called the ID complex. We characterized the FANCI, FANCD2 and FANCI/FANCD2 double knockout cell lines and identified non-overlapping functions in the replication stress response. In fact, we found that only FANCD2 is required for restart of stalled replication forks and FANCI may even inhibit restart when FANCD2 is absent. In addition, FANCD2 has a more vital role in homologous recombination, and FANCI promotes apoptosis in the absence of FANCD2 with replication stress. In Chapter 3 we investigate FANCN, an FA group 3 protein that is associated with more severe FA disease. We used FANCN conditional knockout cells to determine that FANCN is essential for viability and genome stability. In addition, we evaluated FANCN FA-associated mutations and breast cancer-associated variants of unknown significance (VUS) mutations. We confirmed that the BRCA1/2 binding domains of FANCN are not essential for viability and identified two VUS mutations as potentially pathogenic. In conclusion, we have demonstrated alternative functions of FA proteins in response to replication stress as a potential source of FA disease heterogeneity. In addition, we have demonstrated that FANCN is essential for viability whereas FANCI and FANCD2 are not, providing insight into both the frequency of occurrence and severity of FA disease associated with these different genes. Finally, we created isogenic cell lines that are a valuable asset for the FA and breast cancer fields for further investigations into protein function, characterization of patient mutations, and screening novel therapeutics.