High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.
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 degrees 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 polypeptides are 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.
Juodka
B
B
Department of Biochemistry and Biophysics, Vilnius University, Lithuania.
Spiess
E
E
Angiolillo
A
A
Joswig
G
G
Rothbarth
K
K
Werner
D
D
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D012965:Sodium Chloride; D012967:Sodium Dodecyl Sulfate; D013045:Species Specificity
D004268:DNA-Binding Proteins; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D004247:DNA; D011494:Protein Kinases; D000251:Adenosine Triphosphatases