DNA nucleotide excision repair-dependent signaling to checkpoint activation.
Eukaryotic cells respond to a variety of DNA insults by triggering a common signal transduction cascade, known as checkpoint response, which temporarily halts cell-cycle progression. Although the main players involved in the cascade have been identified, there is still uncertainty about the nature of the structures that activate these surveillance mechanisms. To understand the role of nucleotide excision repair (NER) in checkpoint activation, we analyzed the UV-induced phosphorylation of the key checkpoint proteins Chk1 and p53, in primary fibroblasts from patients with xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), or UV light-sensitive syndrome. These disorders are due to defects in transcription-coupled NER (TC-NER) and/or global genome NER (GG-NER), the NER subpathways repairing the transcribed strand of active genes or the rest of the genome, respectively. We show here that in G0/G1 and G2/M phases of the cell cycle, triggering of the DNA damage cascade requires recognition and processing of the lesions by the GG-NER. Loss of TC-NER does not affect checkpoint activation. Mutations in XPD, XPB, and in TTDA, encoding subunits of the TFIIH complex, involved in both transcription and NER, impair checkpoint triggering. The only exception is represented by mutations in XPD, resulting in combined features of XP and CS (XP/CS) that lead to activation of the checkpoint cascade after UV radiation. Inhibition of RNA polymerase II transcription significantly reduces the phosphorylation of key checkpoint factors in XP/CS fibroblasts on exposure to UV damage.
Marini
Federica
F
Dipartimento di Scienze Biomolecolari e Biotecnologie, Universitá degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. federica.marini@unimi.it
Nardo
Tiziana
T
Giannattasio
Michele
M
Minuzzo
Mario
M
Stefanini
Miria
M
Plevani
Paolo
P
Muzi Falconi
Marco
M
D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D005347:Fibroblasts; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000235:genetics; D014983:Xeroderma Pigmentosum / Q000235:genetics
D004247:DNA