Searching for potential Z-DNA in genomic Escherichia coli DNA

The Clarke-Carbon library with Escherichia coli DNA cloned into plasmid ColE1 was partially screened for Z-DNA with the monoclonal antibody Z-D11 using the retardation of the covalently closed circular DNA-protein complex by nitrocellulose filters. About 85% of the plasmids tested at "natural" supercoil density bound to the filter. Together with binding studies of the iodinated antibody, one Z-DNA segment per about 18,000 base-pairs of E. coli DNA is observed. One clone containing the region around the lactose operon, pLC20-30, was studied in detail. Subcloning a partial Sau3A digest and selection with antibodies gave three different Z-forming sites. They were mapped to within about +/- 20 base-pairs by preparing unidirectional deletion clones, selection of protein binding plasmids on nitrocellulose filters and subsequent sizing on agarose gels. The size of the Z-DNA-forming segments was estimated from two-dimensional gels of topoisomer mixtures. Together with results from sequencing of the plasmid DNA using exonuclease III to create single-stranded templates, stretches of alternating purine-pyrimidine tracts of 12 to 15 base-pairs were found to be responsible for Z-DNA formation. One of the sites was found in the middle of the lacZ gene, where it might be an obstacle for RNA polymerase. The methods used here should also be helpful for studying other DNA-protein sites, especially if they exist only in supercoiled DNA.