Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cell nuclear antigen.
The cyclin-dependent kinase inhibitor p21CDKN1A plays a fundamental role in the DNA-damage response by inducing cell-cycle arrest, and by inhibiting DNA replication through association with the proliferating cell nuclear antigen (PCNA). However, the role of such an interaction in DNA repair is poorly understood and controversial. Here, we provide evidence that a pool of p21 protein is rapidly recruited to UV-induced DNA-damage sites, where it colocalises with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase delta, XPG and CAF-1. In vivo imaging and confocal fluorescence microscopy analysis of cells coexpressing p21 and PCNA fused to green or red fluorescent protein (p21-GFP, RFP-PCNA), showed a rapid relocation of both proteins at microirradiated nuclear spots, although dynamic measurements suggested that p21-GFP was recruited with slower kinetics. An exogenously expressed p21 mutant protein unable to bind PCNA neither colocalised, nor coimmunoprecipitated with PCNA after UV irradiation. In NER-deficient XP-A fibroblasts, p21 relocation was greatly delayed, concomitantly with that of PCNA. These results indicate that early recruitment of p21 protein to DNA-damage sites is a NER-related process dependent on interaction with PCNA, thus suggesting a direct involvement of p21 in DNA repair.
Dipartimento di Medicina Sperimentale, sez. Patologia generale, Università di Pavia, 27100 Pavia, Italy.
Paola
P
Perucca
Ornella
O
Cazzalini
Oliver
O
Mortusewicz
Daniela
D
Necchi
Monica
M
Savio
Tiziana
T
Nardo
Lucia A
LA
Stivala
Heinrich
H
Leonhardt
M Cristina
MC
Cardoso
Ennio
E
Prosperi
D000818:Animals; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology* / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D014157:Transcription Factors; D014162:Transfection; D014466:Ultraviolet Rays
D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C087628:TCEAL1 protein, human; D014157:Transcription Factors