Article

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

ABSTRACT

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.
    Full-text · Article · Dec 2013 · The Scientific World Journal
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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.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "The most common UV-induced lesions are 6–4 photoproducts and cyclobutane pyrimidine dimers that are mainly repaired by nucleotide-excision repair (NER). During NER small ssDNA gaps are formed that likely trigger ATR-mediated checkpoint activation (Marini et al., 2006;Warmerdam et al., 2009). Cell cycle regulation of NER, which could explain low RPA focus formation in G2 cells after UV, was studied by incubating UVtreated cells with nucleotide analogue EdU, which is incorporated in newly synthesized DNA in S phase and during NER (Nakazawa et al., 2010) (Fig. 1C; supplementary material Fig. S1D,E). "
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    ABSTRACT: In response to UV light single-stranded DNA intermediates coated by RPA are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment/activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only few single-stranded DNA intermediates are generated. Also, little phosphorylated Chk1 is observed in G2 phase after UV-irradiation, and UV light fails to elicit efficient accumulation of typical DNA damage response proteins at sites of damage in this phase. In contrast, p38 MAPK is phosphorylated in G2 phase cells after UV damage. Interestingly, despite the lack of an obvious activation of the ATR-Chk1 pathway, only the combined inhibition of the ATR- and p38-dependent pathways results in a complete abrogation of the UV-induced G2/M arrest. This suggests that UV light induces less hazardous lesions in G2 phase or lesions created in this phase are less efficiently processed resulting in a low activation of the ATR-Chk1 pathway. UV-induced G2 checkpoint activation in this situation therefore relies on signalling via the p38 MAPK and ATR-Chk1 signalling cascades.
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