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Nucleic Acids Research, 1995, Vol. 23, No. 8 1359-1366
© 1995

MOLECULAR BIOLOGY

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei

Benediktas Juodka, Eberhard Spiess1, Antonella Angiolillo2, Gaby Joswig3, Karsten Rothbarth3 and Dieter Werner3,*

Department of Biochemistry and Biophysics, Vilnius University Vilnius, Lithuania 1Biomedical Ultrastructure Research Unit (0195), German Cancer Research Center PO Box 101949, D-69009 Heidelberg, Germany 2Department of Cell and Molecular Biology, University of Perugia Perugia, Italy 3Division Biochemistry of the Cell (0225), German Cancer Research Center P0 Box 101949, D-69009 Heidelberg, Germany

*To whom correspondence should be addressed

Received December 23, 1994. Revised March 2, 1995. Accepted March 2, 1995.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller,S.A., Dykes,D.D. and Polesky,H.F., Nucleic Acids Res., 16, 1215,1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56{delta}C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptidesare not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 ± 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10–4 M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin depleted nuclei.


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