Investigating the Relationship between Fatty Acid Oxidation and DNA Damage Repair Mechanisms
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Date
2024-05
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The Ohio State University
Abstract
Fatty acid oxidation (FAO) is a key metabolic pathway and a major source of energy for cells and organisms. Metabolic pathways are linked to DNA damage repair, for example FAO can serve as an energy precursor to sustain the cell during the energetically expensive repair process (1). Should DNA face stress conditions or damage, the cell must be able to quickly repair it to retain genome integrity and preserve ability of replication (2). To study how DNA damage is linked to FAO, we use MLE12 cells, a mouse type 2 lung epithelial cell line, with and without deficiency of enzyme carnitine palmitoyl transferase 1A (Cpt1a). Cpt1a is the rate limiting enzyme in the FAO pathway (1, 3). CRISPR modified Cpt1a knock out (Cpt1a KO) and their wild type control, SgCtrl cells were used in a number of experiments, as well as ShRNA Cpt1a knock down (ShCpt1a KD) and their wild type control ShScramble were cultured in HITES media. To induce double strand breaks (DSB) in the DNA, 2mM of Hydroxyurea (HU) was added to the culture media in increments of 30, 60, and 120 minutes, as well as a control (vehicle). DNA damage was analyzed by levels of phosphorylated H2A histone family member X (γH2AX) and accumulation of tumor protein 53 binding protein (53BP1), a scaffolding protein, in both Cpt1a KO and SgCtrl cells. We used RT-qPCR and western blot to analyze the expression level of Cpt1a, and DNA repair enzymes ataxia telangiectasia mutated (Atm), meiotic recombination 11 (Mre11), and breast cancer gene 1 (Brca1) in ShScramble and ShCpt1a KD cells. Mass spectrometry was used to identify amount of acetylation on Histone H4, a histone vital to DNA repair. In addition to this, acetylation activated Atm, Mre11, and phosphorylated tumor protein 53 (p-P53) were selected for investigation of quantity of acetylation on the protein. Lastly, experiments using acetate were performed to restore availability of Acetyl-CoA in the cell in absence of FAO.
We show that Cpt1a deficient cells exhibit increased accumulation of DNA damage in the nucleus at basal conditions and upon HU treatment, most likely due to the down-regulation of repair genes in response to the lack of Cpt1a. Our future studies aim to elucidate the mechanisms that link FAO and the DNA damage repair response.
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Keywords
Fatty Acid, DNA, Metabolism, DNA Damage, Molecular Biology