High-affinity interaction of poly(ADP-ribose) and the human DEK oncoprotein depends upon chain length.
Journal Article (Journal Article)
Poly(ADP-ribose) polymerase-1 (PARP-1) is a molecular DNA damage sensor that catalyzes the synthesis of the complex biopolymer poly(ADP-ribose) (PAR) under consumption of NAD(+). PAR engages in fundamental cellular processes such as DNA metabolism and transcription and interacts noncovalently with specific binding proteins involved in DNA repair and regulation of chromatin structure. A factor implicated in DNA repair and chromatin organization is the DEK oncoprotein, an abundant and conserved constituent of metazoan chromatin, and the only member of its protein class. We have recently demonstrated that DEK, under stress conditions, is covalently modified with PAR by PARP-1, leading to a partial release of DEK into the cytoplasm. Additionally, we have also observed a noncovalent interaction between DEK and PAR, which we detail here. Using sequence alignment, we identify three functional PAR-binding sites in the DEK primary sequence and confirm their functionality in PAR binding studies. Furthermore, we show that the noncovalent binding to DEK is dependent on PAR chain length as revealed by an overlay blot technique and a PAR electrophoretic mobility shift assay. Intriguingly, DEK promotes the formation of a defined complex with a 54mer PAR (K(D) = 6 x 10(-8) M), whereas no specific interaction is detected with a short PAR chain (18mer). In stark contrast to covalent poly(ADP-ribosyl)ation of DEK, the noncovalent interaction does not affect the overall ability of DEK to bind to DNA. Instead the noncovalent interaction interferes with subsequent DNA-dependent multimerization activities of DEK, as seen in South-Western, electrophoretic mobility shift, topology, and aggregation assays. In particular, noncovalent attachment of PAR to DEK promotes the formation of DEK-DEK complexes by competing with DNA binding. This was seen by the reduced affinity of PAR-bound DEK for DNA templates in solution. Taken together, our findings deepen the molecular understanding of the DEK-PAR interplay and support the existence of a cellular "PAR code" represented by PAR chain length.
- Fahrer, J; Popp, O; Malanga, M; Beneke, S; Markovitz, DM; Ferrando-May, E; Bürkle, A; Kappes, F
- August 2010
Volume / Issue
- 49 / 33
Start / End Page
- 7119 - 7130
Pubmed Central ID
Electronic International Standard Serial Number (EISSN)
International Standard Serial Number (ISSN)
Digital Object Identifier (DOI)