The role of S-S bridges of residual protein in the structural organization of DNA is investigated. The effects of various S-S splitting agents on the naturally occurring DNA-RP protein complexes isolated from various eukaryotic and prokaryotic cells are studied. It is demonstrated that, depending on the incubation conditions, thiols induce dissociation of the DNA-RP complexes to double-strand fragment-DNA subunits of varied size. It is found that the DNA-RP complexes contain specific S-S bonds that may determine different levels of DNA organization in the chromosome.Key Words: thiols; protein S-S bridges; DNA structure Investigation of the residual protein (RP) that remains firmly bound to DNA irrespective of isolation technique and produces DNA-polypeptide complexes is important for a better understanding of the structural-functional organization of DNA in the eukaryotic chromosome [18,21,22]. These DNA-polypeptide complexes display site-specific localization in the eukaryotic genome [22]. Of particular importance in this context is the evidence that S-S bridges of RP covalently bound to DNA are involved in the tandem organization of the DNA subunits in the chromosome [14,15,17] and ha the attachment of the replicon loops to the nuclear matrix [3,16], i.e., in a higher level of DNA organization. This paper summarizes the results of our previous studies [5,6,9,19] on pH-dependent thiol-induced fragmentation of naturally occurring eukaryotic and prokaryotic DNA-RP complexes, on the determination of the size of thiol-induced DNA subunits and their secondary structure, and on the effects of thiols on the RP composition in these complexes.
MATERIALS AND METHODSTwo DNA fractions -a water-soluble supramolecular DNA complex (SM DNA) and water-insoluble DNA of the phenol nuclear matrix (PNM DNA) -were isolated by the phenol method from eukaryotic cells (rat liver and thymus, loach sperm cells and erythrocytes; hen erythrocytes, P 388 leukemia cell line) and T4 phage. The isolation procedures for SM DNA and PNM DNA, the conditions for incubation with mercaptoethanol (ME), dithiothreitol (DTT), glutathione reductase, and sodium borohydride, as well as electrophoresis of proteins, elastoviscosimetry, sedimentation, and melting of DNA were described in detail elsewhere [5][6][7].
RESULTSSM DNA contains 1-3% immunogenic RP consisting 30-50% of acid amino acids (predominance of glutamic acid) and actively incorporating 35S-methionine [8]. ELectrophoresis [7] showed that eukaryotic SM DNA is characterized by the presence of a protein quartet in the 50-70 kD region and by the presence of several polypeptides with a molecular weight of 20-40 kD. The protein quartet is resistant to RNAase A, phospholipase C,
