γH2AX in the S Phase after UV Irradiation Corresponds to DNA Replication and Does Not Report on the Extent of DNA Damage

Abstract

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX – the phosphorylated form of a histone variant H2AX – in the S phase within an asynchronous population of cells. γH2AX is often considered as a definitive marker of DNA damage inside a cell. In this report we show that γH2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double strand breaks or in the inhibition of global transcription. Instead γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of γH2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage but the response elicited which peaks in the S phase upon UV irradiation.

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

“…A previous study demonstrated that depletion of SAF-A increased the proportion of cells showing diffuse localisation of the histone variant H2AX phosphorylated at its C-terminus (called γ-H2AX) (Nozawa et al, 2017). Although γ-H2AX has been commonly used as a DNA damage marker, recent studies suggest that diffuse localisation of γ-H2AX within the nucleus is indicative of replication stress rather than DNA damage, whereas a focal γ-H2AX localisation pattern represents DNA damage (Dhuppar et al, 2020; Moeglin et al, 2019). We assessed the impact of depletion of SAF-A with or without replication stress based on γ-H2AX localisation pattern.…”

β-Catenin-Specific Inhibitor, iCRT14, Promotes BoHV-1 Infection-Induced DNA Damage in Human A549 Lung Adenocarcinoma Cells by Enhancing Viral Protein Expression

“…Exposure to various genotoxic stresses, such as UV treatment, leads to a sharp peak of γH2AX and accumulation of γH2AX at the sites of damaged DNA that forms highlighted foci with IFA assay. γH2AX foci or γH2AX protein levels are widely detected to measure DNA damage [7]. Mechanically, the DNA damage due to BoHV-1 infection is largely different from that induced by exposure to canonical genotoxic reagents (such as UV exposure) because viral products, such as viral proteins and viral nucleic acid, are actively introduced into the nucleus.…”

“…In response to DSBs induced by many toxic elements, such as X-rays, γ-radiation, and UV-light irradiation, γH2AX levels increase and accumulate in the DSBs to develop specific foci under fluorescence microscopy, which are generally considered molecular markers of DNA damage [7]. Notably, whether these indicators are also suited for the analysis of BoHV-1 infection-induced DNA damage in A549 cells has not been validated.…”

“…Phosphorylation of the variant histone H2AX at Serine139 (γH2AX) is an early cellular response to the induction of DSBs. Detection of γH2AX is often considered a sensitive molecular marker for monitoring DNA damage and repair [7]. In addition, an alkaline version of a comet assay is also a sensitive method to assess DSBs in individual cells [8].…”

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis

“…Moreover, we found that H4 K16 acetylation, a histone mark that typically accompanies DNA damage response, occurs at low to undetectable levels in basophilic erythroblasts, and we have otherwise not found evidence for higher levels of DNA damage at this stage [ 39 , 43 ]. Alternatively, a recent study has suggested that γ-H2A.X marks active replication forks; thus, high levels of γ-H2A.X could simply reflect higher replication rates and/or a higher proportion of cells in S phase [ 51 , 52 ].…”