Abstract
We have investigated the molecular mechanism by which the proto-oncogene protein DEK, an abundant chromatin-associated protein, changes the topology of DNA in chromatin in vitro. Band-shift assays and electron microscopy revealed that DEK induces both intra- and intermolecular interactions between DNA molecules. Binding of the DEK protein introduces constrained positive supercoils both into protein-free DNA and into DNA in chromatin. The induced change in topology is reversible after removal of the DEK protein. As shown by sedimentation analysis and electron microscopy, the DEK-induced positive supercoiling causes distinct structural changes of DNA and chromatin. The observed direct effects of DEK on chromatin folding help to understand the function that this major chromatin protein performs in the nucleus.
Highlights
Introduction of Positive Supercoils by theDEK ProteinDNA complexes.To determine whether DEK is able to bind different topological forms of DNA, we compared the binding of the DEK protein to supercoiled form I DNA, relaxed form II DNA, and linear form III DNA (Fig. 1B)
In our initial experiments, we failed to detect DEK-induced changes of the topology of relaxed protein-free DNA and concluded that DEK acts in a chromatin-specific manner [6]
DNA or chromatin was incubated with increasing amounts of the DEK protein, and the resulting nucleoprotein complexes were separated by agarose gel electrophoresis (Fig. 1A)
Summary
Purification of GST-DEK—The DEK gene was cloned in the pGEX3 vector (Amersham Biosciences) to generate a GST1 fusion protein. DEK Assay—Purified GST-DEK protein was dialyzed on Whatman filters (Type VS, pore size, 0.025 m) against buffer A-100 (20 mM HEPES at pH 7.6, 100 mM NaCl, 10 mM sodium bisulfite, 1 mM EDTA) in the presence of 1 g/l bovine serum albumin (New England BioLabs) for 90 min at 4 °C. After Proteinase K digestion, DNA was precipitated and analyzed on 0.8% agarose gels in 0.5ϫ TBE (45 mM Tris-borate, 1 mM EDTA) at 2 V/cm for 16 h. Gel Mobility Shift Assays—500 ng of SV40 DNA (forms I, II, and III) or SV40 minichromosomes was incubated with increasing amounts of GST-DEK for 1 h at 37 °C. Purification of topoisomerase I was done from a 500-ml liquid culture exactly as described previously [21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
