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)n 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)n 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)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, 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)4-6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.

Conditions favoring left-handed Z-DNA such as high salinity (> 4 M), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG) n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG) 5 -containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5␣ with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.DNA is a flexible and dynamic molecule which can adopt a variety of conformations (1). Under certain conditions, some DNA sequences can change from the most prevalent righthanded B-DNA conformation and adopt unusual DNA conformations, such as left-handed Z-DNA (2), as well as triplex (3, 4), tetraplex (5), and cruciform structures (6). With increasing salt concentration, inversion of the circular dichroism spectrum of poly-d(CG) nucleotides was observed (7), suggesting the occurrence of an unusual DNA form. However, detailed x-ray crystallographic analysis of d(CG) hexamer was required to established the formation of left-handed Z-DNA (2). Spectroscopic, chemical, and enzymatic analyses have subsequently been used to study the conditions required for Z-DNA formation in vitro (2,8,9). In our previous study (10), the factors required for Z-DNA formation were systematically examined by Z-DNA conformational analysis of a series of short alternating d(CG) sequences cloned in plasmids. The results showed that d(CG) 4 -7 sequences are in a dynamic equilibrium between B-and Z-DNA forms, while longer alternating d(CG) sequences are predominantly in the Z-form. DNA supercoiling was essential ...

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