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A Human-Optimized Damage Suppressor Protein as a Potential Tool to Improve DNA Damage Protection in Human Cells

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The tardigrade’s ability to survive in extreme environments intrigues the scientific community. The mechanistic basis for its durable physiology was partially elucidated with the identification of the tardigrade-specific damage suppressor (Dsup) protein, which is a nucleosome-binding factor that protects against DNA damage when expressed in human cells. Because Dsup is only present in certain tardigrades species, however, it is not optimized to function in human cells. Therefore, this study was conducted to determine if a modified Dsup protein containing the nucleosome binding domain (NBD) of the human high mobility group nucleosomal binding domain 2 (HMGN2) protein could improve protection of human cells against reactive oxygen species (ROS), such as hydroxyl radicals, which damage DNA. Two Dsup-HMGN2 hybrid proteins were designed: Dsup containing the entire NBD of human HMGN2 (Dsup-NBD), and Dsup containing only the core NBD region of HMGN2 (Dsup-coreNBD). Dsup wildtype and the two Dsup-HMGN2 hybrids were integrated into the genome of MCF10A cells (human non-tumorigenic epithelial cells), via homology directed repair (HDR) using the CRISPR/Cas9 system. Following exposure to hydrogen peroxide (which generates hydroxyl radicals), the amount of DNA damage in Dsup containing cells was quantified using the alkaline comet assay. Under the conditions of my experiment, I observed significantly more DNA damage in Dsup-expressing cells when compared to the wildtype MCF10A cells lacking Dsup. Despite this conclusion, the optimization of the Dsup protein for DNA damage protection in human cells could have therapeutic potential when used in conjunction with existing technologies such as gene therapy.

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This item is under embargo until April 6, 2025.