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 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.
Highlights
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
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
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
Summary
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. 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. halobium 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
