To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG) sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG) to d(CG), with 20% of d(CG) and 90% of d(CG) in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG) and d(CG) sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG) or longer sequences, indicating that shorter d(CG) sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG) was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG) sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG) sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.

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