UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells.
DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.
DNA Repair Section, Dermatology Branch, CCR, National Cancer Institute, Bethesda, MD 20892, USA.
Kyu-Seon
KS
Oh
Michael
M
Bustin
Sharlyn J
SJ
Mazur
Ettore
E
Appella
Kenneth H
KH
Kraemer
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000528:radiation effects; D002460:Cell Line; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000528:radiation effects; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D019084:Fluorescent Antibody Technique, Indirect; D006657:Histones / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism* / Q000528:radiation effects; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000528:radiation effects; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C112156:NBN protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; C000607468:Ppm1d protein, mouse; D000071636:Protein Phosphatase 2C