DNA nucleotide excision repair-dependent signaling to checkpoint activation

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universitá degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2006; 103(46):17325-30. DOI: 10.1073/pnas.0605446103
Source: PubMed


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.

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    • "NER comprises two subpathways: the global genome NER and the transcription coupled NER [16–18]. Global genome NER recognises and repairs UV-induced DNA lesions in nontranscribed DNA throughout the genome, while transcription-coupled NER is initiated by damaged DNA-induced arrest of transcribing RNA-polymerase II on the transcribed strand of an active gene [18]. "
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    ABSTRACT: Xeroderma pigmentosum (XP) is a hereditary autosomal recessive disorder characterized by photo hypersensitivity of sun exposed tissues and subsequent several-fold increased risk for malignant changes resulting from impaired ability to repair UV-induced DNA damage. Estimated incidences vary from 1 in 20,000 in Japan to 1 in 250,000 in the USA, and approximately 2.3 per million live births in Western Europe. Diagnosis is made clinically by the presence of unusual sunburns or lentiginosis or onset of cancers at an early age. It is confirmed by cellular tests for defective DNA repair. Although there is no cure for XP as of now, skin problems can be ameliorated with the use of sunscreens, sun avoidance methods, and recurrent tumor excisions. Oral isotretinoin and topical application of 5-fluorouracil to treat actinic keratoses are other therapeutic options. T4N5 and photolyase liposomal lotions are innovations in the therapy of XP. Genetic counselling implicating the effect of consanguineous marriages should be considered in the management of XP patients.
    The Scientific World Journal 12/2013; 2013(2):534752. DOI:10.1155/2013/534752 · 1.73 Impact Factor
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    • "Once the DNA damage is detected by GGR or TCR, the remaining members of the NER process are recruited. Briefly, this process involves unwinding of the DNA helix around the lesion by the helicases XPB and XPD, incision of the DNA upstream and downstream of the lesion by the endonucleases XPF/ERCC1 and XPG and DNA resynthesis and ligation by DNA polymerases δ and ε and DNA ligase I [8]. "
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    ABSTRACT: Nucleotide excision repair (NER) orchestrates the repair of helix distorting DNA damage, induced by both ultraviolet radiation (UVR) and cisplatin. There is evidence that the global genome repair (GGR) arm of NER is dysfunctional in melanoma and it is known to have limited induction in melanoma cell lines after cisplatin treatment. The aims of this study were to examine mRNA transcript levels of regulators of GGR and to investigate the downstream effect on global transcript expression in melanoma cell lines after cisplatin treatment and in melanoma tumours. The GGR regulators, BRCA1 and PCNA, were induced in melanocytes after cisplatin, but not in melanoma cell lines. Transcripts associated with BRCA1, BRCA2, ATM and CHEK2 showed altered expression in melanoma cell lines after cisplatin treatment. In melanoma tumour tissue BRCA1 transcript expression correlated with poor survival and XPB expression correlated with solar elastosis levels. Taken together, these findings provide evidence of the mechanisms underlying NER deficiency in melanoma.
    PLoS ONE 08/2013; 8(8):e70424. DOI:10.1371/journal.pone.0070424 · 3.23 Impact Factor
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    • "UV-induced DNA damage blocks DNA replication fork progression and leads to the recruitment of ATR and its activation [36]. ATR is also activated in G1 phase during the process of NER, when the UV-induced photoproducts are removed and a single-stranded region is formed [37], [38], [44]. ATR activation is enhanced by the action of Exo1, which produces larger ssDNA gaps [45], [46]. "
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    ABSTRACT: The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4(Cdt2) for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.
    PLoS ONE 09/2012; 7(9):e46480. DOI:10.1371/journal.pone.0046480 · 3.23 Impact Factor
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