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DNA Repair During Aging
Published in Alvaro Macieira-Coelho, Molecular Basis of Aging, 2017
What happens when the DNA polymerase, during the course of replication, meets a bulky DNA damage? If one of the repair systems just discussed has not completed the repair before DNA synthesis, replication may be attempted on a damaged template. If the changes in DNA are small (e.g., the presence of an O6-alkylguanine or an apurinic site), miscoding may take place. If the changes are bulky ones (e.g., a pyrimidine dimer), then the replication fork may stop, at least temporarily, near the site of damage. If the fork passes the damage, a gap is left in the Okazaki fragment that is blocked, which leaves a gap in the newly synthesized strand. The gaps are eventually filled in by a mechanism called postreplication repair which is not clearly understood for eukaryotic systems. One model was presented by Villani et al.53 DNA polymerase idles at a damaged site, inserting nucleotides opposite the damage in the new strand, but then removing them by its proofreading exonuclease. The enzyme works according to two mechanisms and continues replication after an arrest. Firstly, DNA synthesis is reinitiated downstream of the block. This results in gaps within the daughter strands which are filled in afterwards. Secondly, after an initial arrest, replication runs past the damage continuously, causing error-prone DNA synthesis. Both mechanisms improve cell survival.53
Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophages
Published in Nanotoxicology, 2020
Célia Ventura, Joana F. S. Pereira, Paulo Matos, Bárbara Marques, Peter Jordan, António Sousa-Uva, Maria João Silva
In agreement with the comet assay results obtained, low levels, or no induction, of DNA strand breaks were reported in A549 cells, BEAS-2B cells and MeT-5A mesothelial cells after exposure to MWCNT-7 and other MWCNT (Lindberg et al. 2013; Louro et al. 2016). Other authors have demonstrated that MWCNT similar to MWCNT-7 can induce high levels of intracellular ROS in Met-5A cells (Yu et al. 2016), V79 cells (Rubio et al. 2016), and A549 cells (Ye et al. 2009; Srivastava et al. 2009), which may be related to MWCNT metal contamination (Pulskamp, Diabaté, and Krug 2007; Visalli et al. 2015, 2017; Tabei et al. 2019). Vales et al. (2016) also reported high levels of intracellular ROS after exposure to the MWCNT NM403 in contrast with its lack of ability to induce oxidative DNA damage detected by the comet assay. The persistency of these DNA lesions might be short and the usual default treatment time of the comet assay allows them to be removed, repaired or leads to cell death, as observed by El Yamani et al. (2017). Hiraku et al. (2015) revealed that MWCNTs induced 8-nitroG formation and NO generation rather than 8-oxoG adducts in A549 cells. Given that the glycosidic bond between 8-nitroG and deoxyribose is chemically unstable and thus 8-nitroG can be spontaneously released to form an apurinic site (Hiraku et al. 2015), it is likely that the resultant DNA strand breaks could be measured by the comet assay. Furthermore, the NO might mediate formation of both oxidized purines and oxidized pyrimidines. The former are recognized by the FpG enzyme and are converted into DNA strand breaks that could be detected by the FPG-modified comet assay (Tretyakova, Wishnok, and Tannenbaum 2000). On the other hand, given that crocidolite genotoxic mode of action is mainly through ROS generation, via the iron-catalysed reduction of oxygen (Fenton-like reaction), mitochondria-derived ROS, and ROS release from inflammatory cells (Srivastava et al. 2009, 2011; Liu, Cheresh, and Kamp 2013), the positive comet assay results were expected. Moreover, the addition of FpG, a DNA repair enzyme that converts oxidative DNA lesions into DNA breaks, raised the level of DNA breaks in monocultured but not in cocultured cells, which suggests that macrophages are not their main contributors. This finding agrees with previous reports that the long length of crocidolite fibers results in the secretion of inflammatory cytokines, but not in a strong ROS production by the macrophages (Padmore et al. 2017).