Cyclobutane Pyrimidine Dimer-specific DNA Glycosylases
Background and Previous Work
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.
Most Recent Work
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.
Ryabinina OP, Minko IG, Lasarev MR, McCullough AK, Lloyd RS. Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase. DNA Repair (Amst). 10(10):1014-22, 2011. PMID: 21889915
Johnson JL, Lowell BC, Ryabinina OP, Lloyd RS, McCullough AK. TAT-Mediated Delivery of a DNA Repair Enzyme to Skin Cells Rapidly Initiates Repair of UV-Induced DNA Damage. J Invest Dermatol. 131(3):753-61, 2011. PMID: 20927123
Golan G, Zharkov DO, Grollman AP, Dodson ML, McCullough AK, Lloyd RS, Shoham G. Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA. J Mol Biol. 362(2):241-58, 2006. PMID: 16916523
Cho YJ, Wang H, Kozekov ID, Kozekova A, Kurtz AJ, Jacob J, Voehler M, Smith J, Harris TM, Rizzo CJ, Lloyd RS, Stone MP. Orientation of the crotonaldehyde-derived N2-[3-Oxo-1(S)-methyl-propyl]-dG DNA adduct hinders interstrand cross-link formation in the 5'-CpG-3' sequence. Chem Res Toxicol. 19(8):1019-29, 2006. PMID: 16918240
Walker RK, McCullough AK, Lloyd RS. Uncoupling of nucleotide flipping and DNA bending by the T4 pyrimidine dimer DNA glycosylase. Biochemistry. 45(47):14192-200, 2006. PMID: 17115714
Harbut MB, Meador M, Dodson ML, Lloyd RS. Modulation of the turnover of formamidopyrimidine DNA glycosylase. Biochemistry. 45(23):7341-6, 2006. PMID: 16752923
Lloyd RS. Investigations of pyrimidine dimer glycosylases--a paradigm for DNA base excision repair enzymology. Mutat Res. 577: 77-91, 2005. PMID: 15923014