Changes in the DNase I sensitivity of DNA sequences within the yeast 2 μm plasmid nucleoprotein complex effected by plasmid-encoded products

Abstract

We examined the effect of plasmid-encoded gene products on two DNase-I-sensitive regions of DNA in the yeast 2 μm plasmid nucleoprotein complex. For these studies, each sensitive region was cloned into an appropriate vector, and the chimeric plasmids were transformed into yeast. Nucleoprotein complexes of the chimeric plasmids were partially purified and tested for sensitivity to DNase I digestion. One sensitive region is between the 3′ end of the 2 μm plasmid coding region D and the plasmid REP3 locus. This region was more sensitive and exhibited a different cleavage pattern when purified from a yeast strain containing endogenous 2 μm plasmid copies than when purified from a yeast strain lacking plasmid copies. Examination of the effect of individual gene products and combinations of the various gene products revealed that the plasmid's REP1, REP2 and D loci were all necessary to restore the pattern to that found in the preparation containing endogenous 2 μm plasmid copies. The other sensitive region studied brackets the binding site of the plasmid-encoded FLP protein, which catalyzes site-specific recombination between the 2 μm plasmid's inverted repeated sequences. In contrast to the first sensitive region examined, the sensitive region in the inverted repeat was less sensitive in chimeric plasmids isolated from a yeast strain containing endogenous 2 μm plasmid copies than from one lacking endogenous copies. Presumably, this protection results from the binding of the FLP protein.