When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.

Toxicological Comparison of Mancozeb and Zoxamide Fungicides at Environmentally Relevant Concentrations by an In Vitro Approach

Mancozeb (MZ) and zoxamide (ZOX) are fungicides commonly used in pest control programs to protect vineyards. Their toxic and genotoxic potential were investigated in vitro on HepG2 and A549 cell lines at environmentally relevant concentrations. Cytotoxicity, apoptosis, necrosis and intracellular reactive oxygen species (ROS), comet assay and a micronucleus test with CREST immunofluorescence were used. The expression of a panel of genes involved in apoptosis/necrosis (BAX/BCL2), oxidative stress (NRF2), drug metabolism (CYP1A1) and DNA repair (ERCC1/OGG1) was evaluated by real-time PCR. Both fungicides were cytotoxic at the highest tested concentrations (295.7 and 463.4 µM, respectively); MZ induced necrosis, ZOX did not increase apoptosis but modulated BAX and BCL2 expression, suggesting a different mechanism. Both compounds did not increase ROS, but the induction of CYP1A1 and NRF2 expression supported a pro-oxidant mechanism. The comet assay evidenced MZ genotoxicity, whereas no DNA damage due to ZOX treatment was observed. Positive micronuclei were increased in both cell lines treated with MZ and ZOX, supporting their aneugenic potential. ERCC1 and OGG1 were differently modulated, indicating the efficient activation of the nucleotide excision repair system by both fungicides and the inhibition of the base excision repair system by MZ. Overall, MZ confirmed its toxicity and new ZOX-relevant effects were highlighted.

The Similarities between Human Mitochondria and Bacteria in the Context of Structure, Genome, and Base Excision Repair System

Mitochondria emerged from bacterial ancestors during endosymbiosis and are crucial for cellular processes such as energy production and homeostasis, stress responses, cell survival, and more. They are the site of aerobic respiration and adenosine triphosphate (ATP) production in eukaryotes. However, oxidative phosphorylation (OXPHOS) is also the source of reactive oxygen species (ROS), which are both important and dangerous for the cell. Human mitochondria contain mitochondrial DNA (mtDNA), and its integrity may be endangered by the action of ROS. Fortunately, human mitochondria have repair mechanisms that allow protecting mtDNA and repairing lesions that may contribute to the occurrence of mutations. Mutagenesis of the mitochondrial genome may manifest in the form of pathological states such as mitochondrial, neurodegenerative, and/or cardiovascular diseases, premature aging, and cancer. The review describes the mitochondrial structure, genome, and the main mitochondrial repair mechanism (base excision repair (BER)) of oxidative lesions in the context of common features between human mitochondria and bacteria. The authors present a holistic view of the similarities of mitochondria and bacteria to show that bacteria may be an interesting experimental model for studying mitochondrial diseases, especially those where the mechanism of DNA repair is impaired.

The Influence of (5′R)- and (5′S)-5′,8-Cyclo-2′-Deoxyadenosine on UDG and hAPE1 Activity. Tandem Lesions are the Base Excision Repair System’s Nightmare

DNA lesions are formed continuously in each living cell as a result of environmental factors, ionisation radiation, metabolic processes, etc. Most lesions are removed from the genome by the base excision repair system (BER). The activation of the BER protein cascade starts with DNA damage recognition by glycosylases. Uracil-DNA glycosylase (UDG) is one of the most evolutionary preserved glycosylases which remove the frequently occurring 2′-deoxyuridine from single (ss) and double-stranded (ds) oligonucleotides. Conversely, the unique tandem lesions (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) are not suitable substrates for BER machinery and are released from the genome by the nucleotide excision repair (NER) system. However, the cyclopurines appearing in a clustered DNA damage structure can influence the BER process of other lesions like dU. In this article, UDG inhibition by 5′S- and 5′R-cdA is shown and discussed in an experimental and theoretical manner. This phenomenon was observed when a tandem lesion appears in single or double-stranded oligonucleotides next to dU, on its 3′-end side. The cdA shift to the 5′-end side of dU in ss-DNA stops this effect in both cdA diastereomers. Surprisingly, in the case of ds-DNA, 5′S-cdA completely blocks uracil excision by UDG. Conversely, 5′R-cdA allows glycosylase for uracil removal, but the subsequently formed apurinic/apyrimidinic (AP) site is not suitable for human AP-site endonuclease 1 (hAPE1) activity. In conclusion, the appearance of the discussed tandem lesion in the structure of single or double-stranded DNA can stop the entire base repair process at its beginning, which due to UDG and hAPE1 inhibition can lead to mutagenesis. On the other hand, the presented results can cast some light on the UDG or hAPE1 inhibitors being used as a potential treatment.