We have studied the uniformity with which the yeast 2 μm plasmid DNA within its nucleoprotein complex is protected from digestion by DNAase I. To probe for relatively unprotected regions, plasmid nucleoprotein complexes were digested with DNAase I to yield a preparation in which approximately half of the circular DNA molecules had been converted to full-length linear molecules. The sites of the double-strand breaks were then mapped in relation to restriction endonuclease sites using end-label probes. The most prominent sensitive sites were found at positions very close to the beginning and end of a 122 base-pair sequence with dyad symmetry located within the 599 base-pair inverted repetition of the plasmid. The sequence is known to be necessary for plasmid site-specific recombination. Other sensitive sites were mapped to the 5′-side of known coding regions. A unique plasmid sequence located to one side of the replication origin was also sensitive to DNAase I digestion yet did not yield discrete cleavage sites. Cleavage of plasmid DNA stripped of proteins did not result in the appearance of distinct fragments as found after cleavage of the same DNA within the nucleoprotein complex. We conclude from these results that, when complexed with proteins, specific plasmid DNA sequences involved in transcription, replication and recombination are more accessible to nuclease digestion.
Bibliographical noteFunding Information:
This work was supported by Sational Science Foundation grant PCM thank <JamesB odley for critically reading the manuscript.