Cellular repair and toxicity determinants of the major oxidative DNA lesion thymine glycol – TG-TOX

Human cell models in which the major components of base excision repair pathway of TG, DNA glycosylases NTHL1 and NEIL1, have been generated using the technology of CRISPR-Cas9.
TG repair kinetics have been measured at different times after exposure of cells to different oxidizing agents such as chemical agents or ionizing radiation. Comet assays, immunodetection of TG using specific antibodies and HPLC-MS/MS have been used to evaluate the amount of TG.
The French team will apply laser microirradiation to induce TG at pre-selected areas of nuclei of living cells 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 project teams shall reveal functional significance of the individual pathways for recognition and processing of TG in the context of living cells.

In order to evaluate the role of different DNA glycosylases in the repair of TG, the German team has established knockout cell lines by genome editing using the CRISPR-Cas9 technology. Knock-outs for NTH1 and NEIL1 as well as double knockouts have been successfully generated and validated by PCR.
Protein extracts from WT and KO clones were further used to perform in vitro enzymatic activity tests on an oligonucleotide substrate containing a synthetic TG. No residual activity was detected in the NTH1 knockout cells, thereby suggesting that NTH1 has a predominant role in the removal of TG in our cell model. The results obtained by the German team using the host cell reactivation assay (HCR) using plasmids containing single TG in defined positions further confirmed a major role of NTH1 is the repair of TG.
TG repair kinetics evaluated at the genomic level after exposure of the cells to oxidative stress performed using comet assays or immunofluorescence approaches did not allow us to conclude due to their low sensitivity. Thus, a collaboration was stablished with Dr. Michael Musheev (IMB, Mainz), a specialist in the detection of DNA damage by the use of the highly sensitive HPLC-MS/MS. The levels of TG in the different cell models generated in this project are under analysis.
We have demonstrated during this project that both NTH1 and NEIL1 are co-recruited to chromatin after exposure of the cells to KBrO3 and that cohesin and Mediator complexes are required for the relocalization. These results represent a major advance in the field by unveiling a new role of cohesin and Mediator complexes, mostly known for their roles in genome organization and transcriptional regulation, in BER of oxidized bases and were published in Nucleic Acid Research in 2020.
The recruitment kinetics of NTH1 and NEIL1 have been further monitored after local damage induced by laser microirradiation in real time in living cells.

During the rest of the project we will continue our efforts to investigate the repair kinetics of TG in the nuclear genome as well as to elucidate the participation of the different DNA repair pathways. For this, the laser microirradiation technique will be further improved by the French partner in order to induce damage in a higher number of cells, required to determine TG excision kinetics by fluorescent microscopy. Those results will be complemented by the measurements performed with HPLC/MS-MS after induction of global DNA damage.
The recruitment of NTH1 and NEIL1 to microirradiation-induced DNA damage will be further characterized by the French team. We will pay particular attention to the impact of mutations affecting the DNA glycosylase activity of the enzymes on the kinetics of recruitment or release to the damaged area.
Despite the major role of NTH1 in the excision of TG, we cannot exclude the participation of NER and NEIL1 in particular contexts. The German team will use the HCR strategy to clarify the roles of NER and NEIL1 by extending the studies to other lesions generated by the same pathway as TG such as OH-dU and HydT. This will need the generation of new plasmids containing the synthetic lesions that will be obtained from commercial sources or through collaboration with chemists that have already been stablished in the context of previous projects.
The German team has developed a very sensitive system based on the plasmids containing TG allowing analysis of mutagenesis potentially induced by the presence of the lesion. The results indicated that synthetic TG does not induce any significant increase of the mutation rate during TLS, nor any measurable reduction of the TLS efficiency. The results thus suggest that mutations and replication fork stalling caused by free radical damage to thymine must be mediated by a product different from TG.

Publications
- Lebraud, E., Pinna, G., Siberchicot, C., Depagne, J., Busso, D., Fantini, D., Irbah, L., Robeska, E., Kratassiouk, G., Ravanat, JL, Epe, B., Radicella, JP, Campalans, A. Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal. Nucleic Acids Res. (2020) doi:10.1093/nar/gkaa611.

Communications in national and international meetings
- Lebraud, E., d’Augustin, O., Dépagne, J., Pinna, G., Radicella, J.P., Campalans, A. New role of chromatin organizers in the recognition and repair of oxidative DNA damage. IMB Conference “Chromosome Territories & Nuclear Architecture” 2019, Mainz. EMBO price for the best poster presentation
- Robeska, E., Lebraud, E. Rodriguez, M., Müller, N., Farag, A., Radicella, J.P., Khobta, A., Campalans, A. Subnuclear Dynamics of Thymine glycol DNA Glycosylases NTH1 and NEIL1 in response to oxidative DNA damage. IMB Conference. “Chromosome Territories & Nuclear Architecture”, 2019, Mainz. (Poster presentation)

- Sarmini, L. and Khobta, A. Nucleotide excision repair of oxidatively induced DNA damage. GUM young scientist workshop, 2021. Award for the best talk

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.