Cellular repair and toxicity determinants of the major oxidative DNA lesion thymine glycol

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Thymine glycol (TG) is a major oxidation product of thymine and 5-methylcytosine in DNA generated by ionizing radiation (IR) and, to a lesser extent, by free radicals of endogenous origin. The non-planar structure of TG and its inability to form Watson-Crick hydrogen bonding result in a significant degree of DNA helix distortion and a substantial capacity to hinder replication of the damaged DNA. The peculiar properties of TG account for its pronounced cytotoxicity as compared to other oxidative base modifications. Biochemical studies demonstrated that at least two DNA glycosylases can initiate base excision repair of TG and the involvement of several additional DNA repair mechanisms is considered plausible. However, the true significance in cells and possible functional overlaps between such repair pathways remain to be explored.The present project is a collaborative effort aimed at elucidating the biological relevance of different repair pathways proposed for TG as well as identifying factors that determine the repair pathway choice in human cells. We will generate human cell models in which the major components of the base excision repair pathway of TG (DNA glycosylases NTHL1 and NEIL1) and of putative alternative repair pathways will be inactivated. The French team will apply laser microirradiation of pre-selected areas of nuclei of living cells in the presence of photosensitizers to detect recruitment of components of the specific DNA repair pathways and to measure repair in situ. The German team will introduce synthetic TG into functional reporter genes and analyse the repair as well as the outcomes of TG for transcription and replication of the damaged template DNA in transfected cells. The complementary approaches of the teams shall reveal the functional significance of the individual pathways for recognition and processing of TG in the context of living cells.