Naturally occurring cross-links in yeast chromosomal DNA

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 x 10⁸ were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of crosslinks in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equillibrium banding in CsCl. Experiments with larger E. coli DNA molecules (Mn = 5.2 x 10⁸) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are a small number of crosslinks, one to four, per molecule.