title: HhaI methylase and restriction endonuclease as probes for B to Z DNA conformational changes in d(GCGC) sequences.
abstract: The capacity of the modification methylase (MHhaI) and restriction endonuclease (HhaI) form Haemophilus 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 (dC-dG) as well as numerous individual d(GCGC) sites distributed around the vector were studied. Negative supercoiling was used to convert the (dC-dG) tracts (approximately 30 bp in length) from a right-handed to a left-handed conformation. (Methyl-3H)-SAM was used to localize and quantitate modified d(GCGC) recognition sites, whereas cleavage by HhaI was used to detect unmethylated sites. In the left-handed Z-form, the (dC-dG) blocks were not methylated by MHhaI and not cleaved by HhaI. A two-dimensional gel analysis of a family of 33 topoisomers treated with MHhaI revealed that the lack of methylation in the (dC-dG) 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 HhaI and MHhaI as probes for different DNA structures and conformational transitions under physiological conditions.
mesh_terms: D001483:Base Sequence; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; D006190:Haemophilus / Q000201:enzymology; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012436:S-Adenosylmethionine / Q000378:metabolism
chemical_list: D012436:S-Adenosylmethionine; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases