Analysis of Left-handed Z-DNA Formation in Short d(CG)n Sequences in Escherichia coli and Halobacterium halobium Plasmids

Abstract

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 rep-licating in either Escherichia coli or the halophilic ar-chaeum 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 Band 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. Experiments in the early 1970s showed that exposure of poly(d(CG)) DNA sequences to high salt concentrations resulted in inversion of the circular dichroism spectrum due to the formation of an unusual DNA structure (1). Subsequently, detailed structural analysis of a d(GC) hexamer in 4 M NaCl by x-ray crystallography (2) showed that the DNA forms a left-handed helical conformation, with alternating glycosidic bonds in anti and syn conformation and zigzag tracking of the phosphate backbone (hence, the name, Z-DNA). Since these initial studies, many other investigations have confirmed the occurrence of left-handed Z-DNA using a variety of approaches, including immunological (3), enzymatic (4), chemical (5), and spectroscopic methods (for review, see Ref. 6). The factors that influence the equilibrium between Band Z-DNA in vitro have also been studied. The nucleotide sequence is important for Z-DNA formation (7-9); Z-DNA is generally adopted by alternating purine-pyrimidine sequences. Among these sequences, the d(CG) repeat has been shown to be the most favorable, the d(CA) repeat is intermediate, and the d(AT) repeat is the least favorable sequence for Z-DNA formation. Negative supercoiling of DNA is also important for Z-DNA formation (4, 10, 11), as the higher energy status of negatively supercoiled DNA may be relieved by opposite handed helix formation. Under high superhelical density, some sequences with imperfect purine-pyrimidine repeats adopt Z-DNA confor-mation (9, 12). The presence of salts and some small molecules also affects Z-DNA formation (1). High salt concentration is thought to stabilize Z-DNA conformation by shielding the repulsion of negatively charged phosphate groups, which are closer in Z-DNA than in B-DNA. Finally, chemical modifications such as methylation (13) and bromination (14) also affect Z-DNA formation due to steric effects. Studies directed at in vivo Z-DNA formation have provided evidence for left-handed DNA in the genomes of a variety of organisms, including plants, animals, and microorganisms (15-22). No studies, however, have been directed at the genome of extremely halophilic microorganisms, for example Halobac-terium halobium, which are classified as archaea and which grow at 3-5 M NaCl. It seemed likely that H. halobium may harbor a significant fraction of Z-DNA in its genome since its cytoplasm contains nearly 5 M salts. Moreover, its genomic DNA has a high GC content, 67%, and therefore, a high statistical likelihood of alternating d(CG) repeat sequences (23). We have also found, as part of this study, that plasmid DNA in H. halobium has a greater linking deficiency (and presumably negative supercoiling) than in Escherichia coli (24). These three factors together suggested that Z-DNA in the H. halo-bium genome may be quite prevalent and perhaps provides significant challenges and opportunities to normal genetic processes. As an initial step in evaluating this hypothesis, we constructed a plasmid series containing short d(CG) repeats and capable of replicating in H. halobium and E. coli, and we systematically studied the importance of d(CG) repeat length, superhelical density, and NaCl concentration in Z-DNA formation. As documented in this report, increasing the length of d(CG) repeats and DNA supercoiling, but surprisingly not salt concentration, were found to promote Z-DNA formation.