Browsing by Subject "Fanconi anemia"

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    The Fanconi anemia pathway and HELQ work alongside dormant replication origins to suppress replication-associated genome instability
    (2014-07) Luebben, Spencer William
    DNA replication is continually impeded by endogenous lesions that cause the stalling of replication forks. If left unchecked, this threatens the integrity of the genome and may be a driver of cancer development. Utilizing the Mcm4chaos3 mouse model, we found that dormant replication origins, which act as backup initiation sites, play a critical role in the recovery of stalled replication forks. A reduced number of dormant origins in these mice led to persistently stalled forks, incomplete replication and the mis-segregation of sister chromatids in mitosis, causing elevated genome instability. Mcm4chaos3/chaos3 cells also displayed intrinsic activation of the Fanconi anemia (FA) pathway, suggesting that it too plays a functional role in fork progression. Indeed, disruption of FA pathway activation in the Mcm4chaos3/chaos3 background led to an even higher number of persistently stalled forks. Furthermore, we discovered that a lack of dormant origins also leads to delayed replication, as seen by extremely late DNA synthesis. Accordingly, concomitant loss of both mechanisms led to heightened genomic instability, causing mice to either die shortly after birth or exhibit accelerated tumorigenesis. Finally, we investigated if HELQ is perhaps another FA gene by characterizing the first Helq mutant mouse model (Helqgt). Helqgt/gt cells/mice displayed modest FA-like phenotypes such as interstrand crosslink hypersensitivity and hypogonadism, but not defects in homologous recombination repair. Rather, HELQ was found to work in parallel to the FA protein FANCC to suppress replication-associated genome instability.
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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.