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

• Applicant: Professor Dr. Andriy Khobta
• Subject Area: Toxicology, Laboratory Medicine ; General Genetics and Functional Genome Biology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406568501

Hydroxyl radical (HO•) – an extremely reactive species arising in cells by radiolysis of water and endogenous redox reactions – causes numerous types of DNA damage. While cellular processing of damage to the sugar phosphate backbone and purine nucleobases has been extensively characterized, relatively little is known about repair and toxic outcomes of damaged pyrimidines. During the first funding period, we have generated gene knockout cell lines and used repair-resistant synthetic nucleotide analogues to investigate repair as well as transcription-blocking and miscoding potential of thymine glycol (TG) – the decay product of thymine (T) and 5-methylcytosine (5mC) induced by their reactions with HO•. The results have established the NTHL1-initiated base excision repair (BER) as the primary TG repair pathway in human cells, in contrast to a negligible or absent role of the nucleotide excision repair (NER) pathway. In addition, we observed a very efficient bypass of the lesion during the gap filling DNA synthesis, with surprisingly low erroneous dNTP incorporation rates, which is hard to reconcile with literature data about cytotoxicity of damage induced by HO•, suggesting that the most harmful adduct in DNA could be different from TG. In fact, HO• addition to pyrimidine bases leads to formation of a radical intermediate, which further decays to either TG or a structurally unrelated 5-hydroxy-methylhydantoin (HydT) lesion. The additional complexity level to the HO• genotoxicity pathway makes us hypothesize that some of adverse effects conventionally assigned to TG may arise from HydT. Even though biological outcomes and repair mechanisms of HydT in mammalian cells are largely unexplored, synthetic HydT lesion as well as its BER-resistant analogue have become available through our collaboration with synthetic chemists during the first funding period. We, therefore, plan to extend the original project scope, further aiming at determination of miscoding properties of both TG and HydT lesions by in-depth analyses of their dNTP incorporation profiles during gap filling DNA synthesis in cells transfected with templates containing synthetic modifications. We also aim to resolve the DNA repair mechanism removing HydT lesion from DNA in the cellular context, as previously done for TG.

• DFG Programme: Research Grants