Browsing by Subject "Base Excision Repair"

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    Clarifying the Role of the CST Complex in DNA Replication and Repair
    (2021-12) Wysong, Brandon Carter; Balakrishnan, Lata; Marrs, James A.; Perrin, Benjamin J.
    Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance via inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates in vitro. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.
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    Inhibition of Ape1's DNA Repair Activity as a Target in Cancer: Identification of Novel Small Molecules that have Translational Potential for Molecularly Targeted Cancer Therapy
    (2009-12) Bapat, Aditi Ajit; Kelley, Mark Richard, 1957-; Georgiadis, Millie M.; Turchi, John J.; Smith, Martin L.
    The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell.