Browsing by Subject "Homologous recombination"

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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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    Repair of DNA-protein crosslinks in mammalian cells
    (2018-07) Chesner, Lisa
    The work below describes a new assay called strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) used to study the repair of DNA-protein crosslinks in mammalian cells. DNA-protein crosslinks (DPCs) are bulky lesions which disrupt important cell processes such as transcription and replication. They are formed by endogenous molecules such as formaldehyde and exogenous damaging agents such as ionizing radiation. However, the repair mechanisms associated with their repair are still unclear. Chapter 1 of this document provides background information on the formation, biological consequences, current models, and methods used to study DPC repair. Chapter 2 describes the SSPE-qPCR assay and its uses/limitations for studying the repair of plasmids containing DPCs or other polymerase-blocking adducts transfected into mammalian cells. Chapter 3 describes results generated using this assay to assess the role of nucleotide excision repair in DPC repair and highlights the versatility of the SSPE-qPCR assay. Chapter 4 extends observations made in Chapter 3 by using SSPE-qPCR to examine repair of DPC-containing plasmids in the presence of a homologous donor. It also provides evidence for homologous recombinational repair of DPCs in mammalian mitochondria. Overall, this work provides additional insight into the mechanisms of DPC repair in the nucleus and mitochondria using a quantitative, flexible assay that has not been available previously.