A minority of foci or pan-nuclear apoptotic staining of gammaH2AX in the S phase after UV damage contain DNA double-strand breaks.
UV irradiation induces histone variant H2AX phosphorylated on serine 139 (gammaH2AX) foci and high levels of pan-nuclear gammaH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of gammaH2AX 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 gammaH2AX response: a minority of gammaH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of gammaH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear gammaH2AX without foci of either gammaH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear gammaH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear gammaH2AX were further increased by loss of the bypass polymerase Pol eta and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced gammaH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear gammaH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of gammaH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.
de Feraudy
Sebastien
S
Department of Dermatology, University of California, San Francisco, CA 94115, USA. sdeferaudy@partners.org
Revet
Ingrid
I
Bezrookove
Vladimir
V
Feeney
Luzviminda
L
Cleaver
James E
JE
D017209:Apoptosis; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002469:Cell Separation; D053903:DNA Breaks, Double-Stranded; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007700:Kinetics; D016196:S Phase; D000071857:Tumor Suppressor p53-Binding Protein 1; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; C468783:H2AX protein, human; C498301:TP53BP1 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases