Abstract
The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly.
Highlights
(GG-nucleotide excision repair (NER)), which removes lesions from the entire genome, and transcription-coupled NER, which eliminates DNA damage from the transcribed strand of the actively transcribed genes [1, 2]
DDB2, which is a subunit of the UV-damaged DNA binding (UV-DDB) complex, is shown to undergo ubiquitin-mediated proteolysis following UV irradiation (19 –21), and the timely degradation of DDB2 has an important role in the arrival of XPC to the damage site in vivo [22]
We have studied the function of p38 MAPK in NER in cells exposed to UV irradiation
Summary
Cell Lines and Treatments—Normal human fibroblasts (OSU-2) were established in our laboratory as described previously [42]. HeLa cells stably transfected with NH2-terminal FLAG-hemagglutinin-tagged DDB2 (HeLa-DDB2) were a gift from Dr Yoshihiro Nakatani (Dana-Farber Cancer Institute, Boston, MA) These cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2. Whole cell lysates were prepared by boiling in SDS lysis buffer (2% SDS, 10% glycerol, 10 mM dithiothreitol, 62 mM Tris-HCl, pH 6.8, and protease inhibitor mixture) for 10 min. The supernatant was diluted with radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixture) and subjected to immunoprecipitation with anti-FLAG M2 affinity gel (Sigma). The digital images were captured with a cooled CCD camera and processed with the help of its SPOT software (Diagnostic Instruments, Sterling Heights, MI)
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