Characterization of CDKN1A (p21) binding to sites of heavy-ion-induced damage: colocalization with proteins involved in DNA repair.
OBJECTIVE
To determine an association of locally accumulated CDKN1A and DNA repair proteins at the sites of heavy-ion traversals.


METHODS
CDKN1A, PCNA, DNA-PK, hMre11 and Rad50 were investigated for their subnuclear localization after irradiation with heavy-ions using immunocytochemical staining and confocal laser-scanning microscopy. Human fibroblasts (normal diploid or XPA, ATM- or NBS1-deficient lines and HPV16 E6-transfected cells) were used.


RESULTS
CDKN1A formed nuclear foci in G0/G1 normal human fibroblasts at the sites of particle traversal. Foci were persistent over hours and vanished after treatment with DNase-I. Formation of foci also occurred in NBS1- or ATM-deficient lines and in cells functionally abrogated for TP53. In normal fibroblasts, CDKN1A foci colocalized with particle-induced foci of the hMre11 and Rad50 proteins. However, only CDKN1A relocalization was observed in irradiated NBS1 cells. PCNA foci temporarily colocalizing with CDKN1A were also detected in normal fibroblasts after exposure to heavy-ions. In contrast, no radiation-induced subnuclear relocalization was found for DNA-PK.


CONCLUSIONS
CDKN1A foci arise rapidly at sites of localized DNA damage induced by heavy-ions and are associated with the chromatin. Evidence is provided that localization of CDKN1A to foci is not dependent on functional TP53 and occurs independently of the formation of the hMre11/Rad50/NBS1 complex. The data support a yet unknown role of CDKN1A in sensing or early processing of radiation-induced DNA lesions.
Jakob
B
B
Gesellschaft für Schwerionenforschung, Biophysik, Planckstrasse 1, D-64291 Darmstadt, Germany.
Scholz
M
M
Taucher-Scholz
G
G
D001665:Binding Sites; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D005656:Fungal Proteins / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D007150:Immunohistochemistry; D007477:Ions; D000076228:MRE11 Homologue Protein; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D007477:Ions; C121766:MRE11 protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011500:Protein Synthesis Inhibitors; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D003513:Cycloheximide; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D003850:Deoxyribonuclease I