Fluorescence-based Approaches for Studying Nucleotide Excision Repair
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Nucleotide excision repair (NER) is a versatile DNA repair pathway known for its wide substrate specificity. NER is able to recognize and remove a variety of helix distorting DNA lesions including those induced by ultraviolet (UV) light, various environmental mutagens and chemotherapeutic agents. The importance of NER is underscored by the inherited human disorder xeroderma pigmentosum (XP), which is caused by the deficiency of genes that are involved in NER. The characteristics of XP patients include an extreme sensitivity to UV light and a more than 2000-fold increased incidence of skin cancer. NER works through a "cut and patch" mechanism that involves the excision of a 24-32mer oligonucleotide containing the damage and restoration the original sequence of the DNA through repair synthesis using the non-damaged strand as the template. NER involves the concerted action of over 30 proteins that work in a sequential order. The rate of repair by NER depends on the degree of duplex destabilization induced by a lesion. Repair rates have been determined using two types of in-vitro NER assays that require the use of radioactive materials to label and detect NER products. The limitations of using of radioactive materials include the short shelf life of substrates, as well as laborious and cumbersome procedures involved in their preparation. More importantly, currently used in-vitro assays are only able to detect NER products, but not the intermediates or unreacted substrates. The work of this thesis was focused on developing innovative NER assays that combine the advantages of the traditionally used in-vitro NER assays, while overcoming the limitation of using radioactive materials. Our approach was to develop two new NER assays using the incorporation of fluorescent tags via oxime formation and click-chemistry to label NER substrates and products. The advantages of using fluorescent labeling include circumventing issues associated with the use of radioactivity, achieving shorter exposure time and longer lifespan of substrates. Our results suggest that click chemistry in particular holds great promise for successfully use in NER assays. These new fluorescent substrates described in this thesis should be valuable tools for studies of the NER pathway since they will allow for the quantitative determination of NER rates and the visualization of various steps of the NER reaction.