A minority of foci or pan-nuclear apoptotic staining of γH2AX in the S phase after UV damage contain DNA double-strand breaks

Abstract

UV irradiation induces histone variant H2AX phosphorylated on serine 139 (γH2AX) foci and high levels of pan-nuclear γH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of γH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of γH2AX response: a minority of γH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of γH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear γH2AX without foci of either γH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear γH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear γH2AX were further increased by loss of the bypass polymerase Pol η and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damagebinding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced γH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear γH2AX, a preapoptotic signal associated with ATM-and JNK-dependent apoptosis during replication. The formation of γH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.T o maintain the integrity of genomic information under threat of DNA damage, cells employ a range of responses including DNA repair, replication bypass, recombination, cell-cycle checkpoints, and senescence or apoptosis (1, 2). These responses are described by the all-embracing concept of the DNA damage response (DDR) (1). DNA double-strand breaks (DSBs) are important but not the only starting lesion for the DDR.In eukaryotic cells, the DDR is rapidly initiated by the PI3K kinases ataxia-telangiectasia and Rad3-related (ATR) and ataxiatelangiectasia mutated kinase (ATM) (3, 4). ATR is recruited to single-stranded DNA regions at stalled replication forks in response to replication stress (5-8); ATM is activated primarily in response to DSBs (3, 9-11). ATR and ATM phosphorylate serine threonine protein kinase and CHK2, respectively, which activate cell-cycle checkpoints and p53, leading to a p53-dependent apoptotic pathway (12). This kinase cascade phosphorylates serine 139 and tyrosine 142 of the histone variant H2AX. After ionizing radiation accumulates in foci, phosphorylated H2AX (γH2AX) serine threonine protein kinase colocalizes with other markers of DSBs (53BP1, pNBS1, MDC1, and Brca1) in immunofluorescent microscopy (IF) (13-16). γH2AX foci are frequently adopted as quantitative markers for DSBs in IF (17, 18). However, the exact correspondence between γH2AX and D...

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

“…S1D), similar to what had been observed following exposure to bleomycin. Unlike cells showing clear repair foci, where γH2AX and 53BP1 mostly colocalize, 53BP1 did not stain in γH2AX pan-stained cells, which is consistent with previous observations (30,32,33). For serotypes 19F and 3, there was a greater frequency of γH2AX-positive cells compared with uninfected cells, although the ability of these serotypes to induce DSB foci was clearly reduced compared with serotype 4 (Fig.…”

A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions

“…8C). Because ␥H2AX is accumulated at the site DNA damages, including DSB and ICL, which cause stalling of replication fork, 53BP1 protein is mainly enriched at sites of DSB (39). Therefore, the PN staining of ␥H2AX without 53BP1 focus observed in the present study is suggested to represent replication fork stalling caused by ICL rather than DSB.…”

“…Our data show that the lack of REV7 function results in accumulation of ␥H2AX without 53BP1 colocalization at S phase, which could represent unresolved replication-fork stalling caused by DNA damages (39). These findings indicated that S phase ICL repair requires Rev7 and suggest that DNA Pol functions downstream of the FA pathway.…”

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

“…HNE Treatment Induces Phosphorylation of Histone H2A.X at Ser-139-Because HNE causes DNA damage, we examined whether HNE treatment caused DNA double strand breaks by monitoring Ser-139 phosphorylation of H2A.X, which is a sensitive marker for the presence of DNA double strand breaks (52)(53)(54). Western blot studies clearly showed a concentrationdependent phosphorylation of H2A.X in HNE-treated HepG2 and Hep3B (Fig.…”