Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation.
Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects*; D004247:DNA / Q000302:isolation & purification; D003852:Deoxyribonucleoproteins / Q000378:metabolism / Q000528:radiation effects*; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000302:isolation & purification; D008970:Molecular Weight; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects*; D013950:Thymus Gland / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D003852:Deoxyribonucleoproteins; D006657:Histones; D009698:Nucleoproteins; D004247:DNA