Structural integrity of the nuclear matrix: differential effects of thiol agents and metal chelators

Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from BHK nuclei by extraction with 2M-NaCl. The matrices were found to impose at least two levels of structural order upon nuclear DNA. From sedimentation studies it was inferred that metal depletion of the salt-extracted nuclei generated matrix structures, which sedimented at significantly lower rates than control matrices. Fluorescence microscopy revealed that the reduced sedimentation rate is a consequence of the increase in the radius of the DNA halo, i.e. the DNA loops emanating from the residual nucleus. Addition of Cu ions to nuclei prior to salt extraction was found to induce contraction of this DNA halo. These results indicate that Cu ions may play an important role in stabilizing one level of DNA folding. When metal depletion had been brought about by thiol agents, a second effect was observed to occur. Within 15 min, salt-extracted nuclei disintegrated, generating irregularly shaped, slowly sedimenting structures. Disintegration only occurred when the full complement of DNA was still attached to the nuclear matrices. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that treatment with thiols did not detectably alter the polypeptide composition of DNA-depleted residual nuclei. Results of these experiments suggest that both metal-protein interactions and disulphide bonds are important in maintaining higher-order structure in the nucleus. A model to account for these observations is discussed.