A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex.
Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.
D002467:Cell Nucleus / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000032:analysis*; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis
D004273:DNA, Neoplasm; D003852:Deoxyribonucleoproteins; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease