Browsing by Subject "Oxidative damage"

Now showing 1 - 4 of 4
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    Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices
    (American Chemical Society, 2021-07-23) Wettasinghe, Ashan P.; Singh, Naveen; Starcher, Colton L.; DiTusa, Chloe C.; Ishak-Boushaki, Zakari; Kahanda, Dimithree; McMullen, Reema; Motea, Edward A.; Slinker, Jason D.; Biochemistry and Molecular Biology, School of Medicine
    Here, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1−) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1− lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies.
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    Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage
    (Elsevier, 2024) Wysong, Brandon C.; Schuck, P. Logan; Sridharan, Madhumita; Carrison, Sophie; Murakami, Yuichihiro; Balakrishnan, Lata; Stewart, Jason A.; Biology, School of Science
    CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.
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    Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage
    (Oxford University Press, 2016-06) Ding, Ning; Bonham, Emily M.; Hannon, Brooke E.; Amick, Thomas R.; Baylin, Stephen B.; O'Hagan, Heather M.; Medical and Molecular Genetics, School of Medicine
    At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein–protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes.
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    Opening Pandora’s jar: a primer on the putative roles of CRMP2 in a panoply of neurodegenerative, sensory and motor neuron, and central disorders
    (Taylor & Francis, 2012) Khanna, Rajesh; Wilson, Sarah M.; Brittain, Joel M.; Weimer, Jill; Sultana, Rukhsana; Butterfield, Allan; Hensley, Kenneth; Pharmacology and Toxicology, School of Medicine
    CRMP2, also known as DPYSL2/DRP2, Unc-33, Ulip or TUC2, is a cytosolic phosphoprotein that mediates axon/dendrite specification and axonal growth. Mapping the CRMP2 interactome has revealed previously unappreciated functions subserved by this protein. Together with its canonical roles in neurite growth and retraction and kinesin-dependent axonal transport, it is now known that CRMP2 interacts with numerous binding partners to affect microtubule dynamics; protein endocytosis and vesicular cycling, synaptic assembly, calcium channel regulation and neurotransmitter release. CRMP2 signaling is regulated by post-translational modifications, including glycosylation, oxidation, proteolysis and phosphorylation; the latter being a fulcrum of CRMP2 functions. Here, the putative roles of CRMP2 in a panoply of neurodegenerative, sensory and motor neuron, and central disorders are discussed and evidence is presented for therapeutic strategies targeting CRMP2 functions.