Cyclobutane Pyrimidine Dimer-specific DNA Glycosylases
Sunlight exposure causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed in the United States annually. Human cells possess only one mechanism (Nucleotide Excision Repair, NER) for repair of ultraviolet light (UV)-induced DNA damage. This mechanism is defective in individuals affected with Xeroderma Pigmentosum (XP) causing them to be ~2000-fold more cancer-prone than the general population. Human cells do have enzymes to complete another mechanism of DNA damage repair, Base Excision Repair (BER), but lack the specific pyrimidine dimer glycosylase (Pdg) needed to initiate BER in response to UV. The exogenous delivery of a photoproduct-specific Pdg protein to human skin has the potential to enhance DNA repair following UV exposure. Ongoing investigations of two such pdgs (T4-Pdg and cv-Pdg) have successfully modified these enzymes to prevent their ability to catalyze cytotoxic double-strand breaks while retaining UV-specific catalytic activities. Concomitant with these studies, these Pdgs have been engineered to contain a nuclear localization sequence (NLS) and a cell membrane permeabilization sequence (TAT from HIV) to enhance cellular uptake and delivery. The Pdgs can be effectively delivered to the nuclei of repair-proficient human keratinocytes and fibroblasts, as well as NER-deficient XP fibroblasts, accelerating DNA repair. Further, Cv-pdg-NLS encapsulated in a lipid-based delivery vehicle can traverse the stratum corneum of a human skin model and localize to the nuclei of basal and suprabasal keratinocytes, where it enhances repair of UV-induced DNA damage. The main objective of our studies is to investigate the efficacy of these novel genetically modified Pdgs to enhance DNA repair and cell survival in a human skin model.
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