Hhalmethylase and restriction endonuclease as probes for B to Z DNA conformational changes in d(GCGC) sequences

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Abstract
The capacity of the modification methylase (M Hha I) and restriction endonuclease ( Hha I) from Haemophilue haemolyticus to methylate and cleave, respectively, recognition sites which are in right-handed B or left-handed Z structures was determined in vitro Plasmids containing tracts of (dCdG) S8 well as numerous individual d(GCGC) sites distributed around the vector were studied. Negative aupercoiling was used to convert the (dC-dc) tracts (˜30bp in length) from a right-handed to a left-handed conformation. (Methyl-was used to localize and quarititate modified d(GCGC) recognition sites, whereas cleavage by Hhat was used to detect unmethylated sites. In the left handed Z-form, the (dc-dc) blocks were not methylated by MHha I and not cleaved by Hha I. A two-dimensional gel analysis of a family of 33 topoisomers treated with M Hha I revealed that the lack of methylation in the (dc-dc) blocks was directly correlated to the supercoil-induced B to Z transition in these segments. These results are significant with respect to enzyme-DNA interactions in general and provide the basis for using Hha I and M Hha I as probes for different DNA structures and conformational transitions under physiological conditions.