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CAK inhibition decreases gene transcription after UV-induced DNA damage.

By Hany H. Arab (364331), Gulzar Wani (364332), Alo Ray (364333), Zubair I. Shah (364334), Qianzheng Zhu (364336) and Altaf A. Wani (364338)

Abstract

<p>(<b>A</b>) Effect of Cdk7 kinase inhibition on the Ser5-phosphorylation of RNAP II. HCT116-Cdk7<sup>as/as</sup> cells were pretreated with 10 µM 1-NMPP1 or DMSO for 14 h, UV irradiated (20 J/m<sup>2</sup>), and then maintained in fresh medium with the same pretreatment composition for the indicated repair period. The cell lysates were analyzed by Western blotting using anti-phospho-RNAP II and anti-phospho-p53 antibodies. Cellular lamin B serves as a loading control. (<b>B</b>) Transcription and transcription recovery measured by Host Cell Reactivation assay with UV-damaged reporter plasmid. HCT116-Cdk7<sup>as/as</sup> cells were first transfected with undamaged or UV-damaged (1000 J/m<sup>2</sup>) reporter plasmid harboring a CMV-driven luciferase gene for 24 h. After transfection, the cells were maintained in fresh medium containing either 1-NMPP1 or DMSO for another 24 h. The cells were then harvested and the cell lysates were assayed for luciferase activity. The data are expressed as percentage of relative luciferase activity from undamaged reporter 24 h after transfection, and the bars show the calculated mean ± S.E. obtained from at least 4 independent experiments. Symbols * and # indicate significant difference (p<0.05) from 24 h-transfected cells and DMSO-treated transfected cells, respectively. (<b>C</b>) Cdk7 inhibition differentially affects transcription and UV-inducible transcription of <i>p53</i>, <i>p21<sup>waf1</sup></i>, <i>DDB2</i> and <i>GAPDH</i> genes. HCT116-Cdk7<sup>as/as</sup> cells were treated as described in (<b>A</b>) and the total RNA was isolated from 1-NMPP1 or DMSO-treated cells. The <i>p53</i>, <i>p21<sup>waf1</sup></i>, <i>DDB2</i> and <i>GAPDH</i> mRNA was detected by real-time RT–PCR assay using gene-specific primers as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011007#s4" target="_blank">Materials and methods</a>’. The levels of individual mRNA transcripts were expressed relative (fold) to DMSO-treated unirradiated cells as control. Bars represent mean (±SD) of three determinations.</p

Topics: Biochemistry, Molecular Biology, inhibition, decreases, transcription, uv-induced, dna
Year: 2013
DOI identifier: 10.1371/journal.pone.0011007.g007
OAI identifier: oai:figshare.com:article/517294
Provided by: FigShare
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