Oxidative DNA Damage and Repair in the Radioresistant Archaeon Thermococcus gammatolerans.

The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2'-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 106 nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/106 nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry indicated that both have a ß-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-ß-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-ß-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation.

Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

Tools and strategies for DNA damage interactome analysis.

DNA is the target of multiple endogenous and exogenous agents generating chemical lesions on the double helix. Cellular DNA damage response pathways rely on a myriad of proteins interacting with DNA alterations. The cartography of this interactome currently includes well known actors of chromatin remodelling, DNA repair or proteins hijacked from their natural functions such as transcription factors. In order to go further into the characterisation of these protein networks, proteomics-based methods began to be used in the early 2000s. The strategies are diverse and include mainly (i) damaged DNA molecules used as targets on protein microarrays, (ii) damaged DNA probes used to trap within complex cellular extracts proteins that are then separated and identified by proteomics, (iii) identification of chromatin- bound proteins after a genotoxic stress, or (iv) identification of proteins associated with other proteins already known to be part of DNA damage interactome. All these approaches have already been performed to find new proteins recognizing oxidised bases, abasic sites, strand breaks or crosslinks generated by anticancer drugs such as nitrogen mustards and platinating agents. Identified interactions are generally confirmed using complementary methods such as electromobility shift assays or surface plasmon resonance. These strategies allowed, for example, demonstration of interactions between cisplatin-DNA crosslinks and PARP-1 or the protein complex PTW/PP. The next challenging step will be to understand the biological repercussions of these newly identified interactions which may help to unravel new mechanisms involved in genetic toxicology, discover new cellular responses to anticancer drugs or identify new biomarkers and therapeutic targets.

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs.

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.