Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication

Abstract

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells

“…NAC is especially useful for increasing cell antioxidant capacity through the production of glutathione, thereby protecting the organism against reactive species (81,82) and dark CDPs (21). Actually, the use of NAC in UVA-irradiated cell cultures results in replication fork stalled rescue in ATM, ATR and homologous recombination-defective cells (47,53,83), as well as DNA protection against fragmentation (81,84). The observations of this work clearly indicate that the UVA-induced redox process damages human cells, especially XP-V, and in ATR/Chk1 pathway activation, since the use of NAC significantly decreased cell sensitivity and DNA damage, and consequently genotoxic stress (cH2AX signal), as well as Chk1 phosphorylation in UVA-irradiated XP-V cells, even in the presence of an ATR inhibitor.…”

Cooperation and interplay between base and nucleotide excision repair pathways: From DNA lesions to proteins

“…Confirming these observations, studies from our lab showed that protein oxidation by UVA irradiation also affects the ability of human cells to replicate their genetic material, probably due to translesion synthesis (TLS) and NER being affected in irradiated XP-V cells (Moreno et al, 2019ab). Curiously, previous work reported that UVAinduced singlet oxygen leads to DNA replication arrest independently of cell cycle checkpoints activation, probably due to a transient decrease of dNTP pool, probably not related to the oxidation of DNA repair proteins (Graindorge et al, 2015). This suggests that UVA light-induced oxidative stress has a greater contribution in impairing proteins that participate in DNA repair and replication pathways than in inducing direct damage to DNA.…”

Differential correlations between changes to glutathione redox state, protein ubiquitination, and stress-inducible HSPA chaperone expression after different types of oxidative stress

“…During this study, cells from passage 6 to 15 were cultured in Bcomplete^DMEM, namely DMEM high glucose (Lonza or Gibco) supplemented with penicillin and streptomycin (100 U ml −1 ), 10% fetal bovine serum, and 2 mM glutamine at 37°C. As indicated in the text, we also used the MRC5Vi cell line that are immortalized human fibroblasts and cultured as previously described (Graindorge et al, 2015). For cell viability experiments, cells were at 40-50% confluent on the day of the treatments, while for all other experiments, the treatments were performed when cells were 80-90% confluent and in serum-free, phenol red-free, and glutamine-free DMEM medium in which stock solutions of the different chemicals were diluted.…”