1887

Abstract

DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat–DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription.

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2011-07-01
2019-10-23
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