Cyclobutane Pyrimidine Dimer-specific DNA Glycosylases

Even though the Pacific Northwest is generally associated with only modest sun exposure, the state ranks in the top 5 nationally for sun-light-induced melanoma skin cancer and other forms of skin cancer are also very prevalent.  Although the reasons underlying these facts are highly complex, it is important to take steps to prevent or at least significantly the onset of these diseases.  Toward this goal, the research laboratories of Drs. Amanda McCullough and Stephen Lloyd have combined research interests and expertise to develop a potential prevention/cure for these skin cancers.  Together, they have patented DNA repair enzymes that when delivered to human skin cells, rapidly repair sunlight-induced DNA damage.  These investigations have recently shown that when human skin is grown in the laboratory, repair of sunlight damage rapidly occurs when the DNA repair lotion is applied.  These studies are leading directly to FDA safety approval and initial human clinical trials.

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.

 

Germane Publications

Lloyd RS. Investigations of pyrimidine dimer glycosylases--a paradigm for DNA base excision repair enzymology. Mutat Res. 577: 77-91, 2005. PMID: 15923014

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

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

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, 753-761 2010. PMID: 20927123 PMCID: 3203208.

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). 10,1014-1022, 2011. PMID: 21889915 PMCID:3185154Dodson ML, Walker RC, Lloyd RS. Carbinolamine formation and dehydration in a DNA repair enzyme active site. PLoS ONE 7(2): e31377, 2012. PMID: 22384015 PMCID: 3285167