Oxidative Stress Induces Telomere Dysfunction and Senescence by Replication Fork Arrest

Abstract

Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.

X-rays Activate Telomeric Homologous Recombination Mediated Repair in Primary Cells

“…Nonetheless, it is well known that ionizing radiation, such as X-rays, principally induce DSBs through an (OS-mediated) indirect mechanism [53]. Several papers in the literature indicated that OS specifically target G-rich regions such as telomeres [54], determining the occurrence of 8-oxo-Guanine (8-oxoG) that in turn increases the rate of stalling of replication forks at telomeres [41,55]. Remarkably, the measurement of the OS was significantly higher in samples exposed to 4Gy of X-rays than in mock-irradiated controls and persisted until day 5 after irradiation.…”

“…Indeed, we do observe a strong reduction in ATRX expression following x-ray irradiation. Another possibility is that the binding of shelterin constituents like TRF2 is transiently disrupted in response to X-rays [41,61,62]. In addition, there is evidence that as telomeres elongate beyond normal limits the binding of TRF1 and TRF2 become diluted [48].…”

“…ChIP analysis was performed as previously described [41]. Briefly, 4 × 10 6 cells were used for each experimental point.…”

The Histone Code of Senescence

“…SIPS is characterized by the accumulation of ROS (reactive oxygen species), due to mitochondrial disfunctions, ER stress or the exogenous administration of oxidative compounds [118][119][120]. Even though SIPS onset is independent from telomere attrition, ROS accumulation can cause telomere disfunction and fusion in primary cells, thus sustaining cell-cycle arrest [120,121].…”

“…SIPS is characterized by the accumulation of ROS (reactive oxygen species), due to mitochondrial disfunctions, ER stress or the exogenous administration of oxidative compounds [118][119][120]. Even though SIPS onset is independent from telomere attrition, ROS accumulation can cause telomere disfunction and fusion in primary cells, thus sustaining cell-cycle arrest [120,121]. Accordingly, murine embryonic fibroblasts (MEFs) cultured in normoxia undergo premature senescence due to the accumulation of ROS-induced DNA damage, while the same cells cultured in hypoxia tend to indefinitely proliferate in virtue of the long telomeres [122].…”