A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease.
T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.
D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000378:metabolism*; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000378:metabolism*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli