The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA.
Binding of exogenous DNA to the nuclear scaffold was investigated using a plasmid DNA (pBR322, EcoRI site deleted) of various topological forms and nuclear subfractions with different levels of nuclear DNA depletion. When supercoiled DNA was incubated with histone-depleted nuclei (nuclear halo), a dose-dependent binding of the DNA occurred, whereas no binding was observed with relaxed and linear forms of DNA. The bound DNA was released upon linearization with BamHI or digestion of the scaffolding structure with proteinase K. Extensive digestion of the halo with micrococcal nuclease generated additional sites which bind both relaxed and linear DNA. In the presence of a large excess of calf thymus DNA, these sites were effectively blocked and the specificity to supercoiled DNA was restored. The binding of all forms of DNA was abolished by heat-denatured DNA. There was no detectable change in linking number of the scaffold-associated supercoils. Competitive binding was observed between supercoiled DNAs with unrelated sequences, indicating that no specific nucleotide sequence is required for the binding. RNA was found to be a weak competitor. A DNA binding assay performed on electrophoretic blots of solubilized nuclear scaffold revealed a protein component with apparent molecular weight of 120,000 which retained selective binding to supercoils. These results suggest that the nuclear scaffold possesses DNA-binding sites for torsionally strained domains of chromatin and that an integral protein factor is involved in the binding. Implications of the findings are discussed in connection with proposed functions of the nuclear scaffold and topoisomerase II.
Tsutsui
K
K
Department of Biochemistry, Okayama University Medical School, Japan.
Tsutsui
K
K
Muller
M T
MT
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D051381:Rats; D011919:Rats, Inbred Strains
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins