Abstract
Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear protein that modifies proteins by forming and attaching to them poly(ADP-ribose) chains. Poly(ADP-ribosyl)ation represents an event of major importance in perturbed cell nuclei and participates in the regulation of fundamental processes including DNA repair and transcription. Although ADPRT serves as a positive cofactor of transcription, initiation of its catalytic activity may cause repression of RNA polymerase II-dependent transcription. It is demonstrated here that ADPRT-dependent silencing of transcription involves ADP-ribosylation of the TATA-binding protein. This modification occurs only if poly(ADP-ribosyl)ation is initiated before TATA-binding protein has bound to DNA and thereby prevents formation of active transcription complexes. Specific DNA binding of other transcription factors including Yin Yang 1, p53, NFkappaB, Sp1, and CREB but not c-Jun or AP-2 is similarly affected. After assembly of transcription complexes initiation of poly(ADP-ribosyl)ation does not influence DNA binding of transcription factors. Accordingly, if bound to DNA, transcription factors are inaccessible to poly(ADP-ribosyl)ation. Thus, poly(ADP-ribosyl)ation prevents binding of transcription factors to DNA, whereas binding to DNA prevents their modification. Considering its ability to detect DNA strand breaks and stimulate DNA repair, it is proposed that ADPRT serves as a molecular switch between transcription and repair of DNA to avoid expression of damaged genes.
Highlights
Participation in DNA repair is regarded as a major function of ADPRT.1 Occurrence of DNA strand breaks induces the enzymatic activity of ADPRT, transfer of ADP-ribose moieties from NADϩ to proteins
It was concluded that this effect is part of a defense mechanism, because DNA repair after ␥-irradiation is stimulated by ADPRT activity (5, 6)
Because it was the goal of the present study to understand how activation of ADPRT may cause repression of transcription, the effect of poly(ADP-ribosyl)ation on transcription factors was analyzed
Summary
Purification of Recombinant Proteins—After overexpression in Escherichia coli cells the human His-tagged ADPRT was purified as described previously (15). In a binding reaction nuclear HeLa extracts (4 g of protein) or recombinant proteins were incubated with 5 ng of 32P-labeled duplex oligonucleotide for 20 min at room temperature in 20 mM Tris-HCl, pH 8, 60 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.05% Nonidet P-40, 10% glycerol, 50 g/ml bovine serum albumin, and 500 ng of poly (dI-dC) in a final volume of 10 l. 20 ng of TBP and 50 ng of TFIIB were incubated with 5 ng of 32P-labeled TATA duplex oligonucleotide for 15 min at room temperature in 20 mM Tris-HCl, pH 8, 80 mM KCl, 10 mM MgCl2, 2 mM dithiothreitol, and 10% glycerol in a final volume of 10 l. All data presented are representative of at least three independent experiments
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