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Nucleic Acids Research, 1985, Vol. 13, No. 17 6137-6154
© 1985

Articles

Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA

Lowell G. Sheflin and David Kowalski*

Department of Cell and Tumor Biology, Roswell Park Memorial Institute Buffalo, NY 14263, USA

*To whom correspondence should be addressed

Received June 24, 1985. Revised August 6, 1985. Accepted August 6, 1985.

Mung bean nuclease was used to probe for recognizable DNA unwinding and unpairing in the plasmid pBR322. In negatively supercoiled DNA, but not relaxed DNA, cleavages occurred preferentially in non-coding regions of the genome. The types of nucleotide sequences cleaved and which non-coding regions were cleaved depended upon environmental conditions. At 37°C, cleavages occurred in an 84 bp A+T–rich sequence in the terminator region of the ampicillin-resistance gene. Recognition is likely based on a novel DNA conformation which occurs in the longest, most dA+dT–rich region of pBR322. In the presence of 1 mM Mg2+, cleavages occurred in inverted repeated sequences in the promoter regions of the RNA primer for DNA replication and ampicillin- and tetracycline-resistance genes as well as the terminator of RNA-1. Potential loops of hairpin (cruciform) structures were cleaved. At 27°C, cleavages occurred near a promoter activated by cAMP receptor protein in vitro and in the 3' non-coding region of the tetracycline-resistance gene. Thus, in supercoiled pBR322 DNA, recognizable DNA unwinding and unpairing occurs preferentially in regulatory regions for transcription and DNA replication.


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