Isolation and Chromosomal Distribution of Natural Z-DNA-forming Sequences in Halobacterium halobium

Abstract

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 ...

Transcriptional profiling of the model Archaeon Halobacteriumsp. NRC-1: responses to changes in salinity and temperature

“…High salt concentrations are also known to convert some DNA sequences, e.g. alternating (CG)-repeats, from the right-handed B to the left-handed Z form, facilitated by negative superhelical stress [19,20]. Organic compatible solutes commonly utilized in the response of bacterial and eukaryotic halophiles to osmotic stress, have also been reported in Halobacterium sp.…”

Periodicities of 10–11 bp as Indicators of the Supercoiled State of Genomic DNA

“…For Halobacterium strains experimental evidence of negative supercoiling has been given earlier. 34,35 This example illustrates that a periodicity analysis of genomic data complements direct experimental studies of DNA superhelicity and histone packaging.…”

“…Interestingly, there are indeed indications of quite specific DNA structures in Halobacterium species including negative supercoiling. 34,35 Figure 8 summarizes the analyses of growth temperatures and periodicities. It can be seen that eubacteria have periods around 11 bp irrespective of their growth temperature.…”

Transcription initiation in Archaea: facts, factors and future aspects

“…The influence of DNA topology on transcription efficiency could be a second mode of global regulation. More than 20 Z‐DNA‐containing genomic fragments have been isolated from H. salinarum making use of an anti‐Z‐DNA antibody (Kim and DasSarma, 1996). If these were located in promoter regions, conditional Z‐DNA formation could influence transcription.…”