Methylation and restriction endonuclease cleavage of linear Z-DNA in the presence of hexamminecobalt (III) ions

Volume 14 Number 18 1986 Nucleic Acids Research Methylation and restriction endonuclease deavage of linear Z-DNA in the presence of nexamminecobalt (III) ions Gerald Soslau1-2, Janet Parker1 and Jeffrey W.Nelson3 Departments of 'Biological Chemistry and ^ematology/Oncology, Hahnemann University, Broad and Vine Streets, Philadelphia, PA 19102 and 'Department of Biology, Leidy Labs, University of Pennsylvania, Philadelphia, PA 19104, USA Received 10 June 1986; Revised and Accepted 27 Augus 1986 INTRODUCTION The left handed helical DNA (Z DNA) structure is readily induced in synthetic alternating dGdC (GC) DNA oligomers and polyners by a variety of salts (1-5) and alcohols (6, 7). The equilibriua between the B and Z DNA foras i3 largely dependent upon the coaposltion and tenperature of the solution and the nucleotlde sequence of the DNA. The effectiveness of cobalt hexamalne cation in stabilizing Z DNA is 5 orders of sagnltude greater than the sodiun ion and 4 orders of magnitude greater than aagnesium (2). This enhanced stabilization has been attributed to increased hydrogen bond foraation between the cobalt cation and the Z DNA surface (8). The B-Z transition has been shown to be influenced by teaperature in the presence of various salts (9-12) and alcohol (10, 13). The theraally-induced transition is both cooperative and reversible and appears associated with structural changes in the solvent nolecules surrounding the DNA (11). Methylation of cytosine in the GC tracts reduces the salt concentration required to induce the B to Z transition and stabilizes the Z form (14, 15). Alternating GC oligoaers inserted into covalently closed circular DNA (cccDNA) can be © IR L Press Limited, Oxford, England. 7237 ABSTRACT These studies employed the synthetic linear DNA, poly dGdC. in the B and cobalt hexanmine chloride (Co (-Induced Z fora to detemine the effect of conformation on protein-DNA Interactions. The rate of the reaction of the restriction endonucleasea, Hha 1 and Cfo I, are reduced with Z DNA as coapared to B DNA. The ability of both restriction endonucleases to react with an aggregate forn of Z DNA (Z* DNA) is found to depend upon how the V DNA Is formed. When Z* DNA is induced by low concentrations of Co (50 uM) , the endonucleases renain active. In the presence of 100 uM Co, which causes increased aggregation, the endonucleases are Inactive. The Hha I DNA Bethyltransferase reacts at equal rates with the B, Z and low cobalt Z* foras and at a greatly reduced rate with the high cobalt Z* forn. These results are significantly different than those observed with Z forn dGdC tracts inserted into circular DNA aolecules. Nucleic Acids Research shifted Into the Z conformation by Increasing the superhellcal density of the molecule (16-18). The negative supercolllng, naturally found In ccc DNA stabilizes the Z DNA conformation (19-21). Localized supercoillng may exist In linear DNA as well, however, It Is difficult to enzymatically regulate as with the topoisomerase treated cccDNA (22). A number of studies clearly Indicate that Z DNA Is not relegated to man-made model systens but also occurs naturally (22, 2 3 ) . While alternating GC sequences aay contribute to natural Z DNA it appears that alternating purlne pyrimidine sequences may be sufficient for Z DNA structures In nature (17, 2 4 ) . The Z DNA structures may be associated with to condense nore readily than B DNA (4, 7, 25, 2 6 ) . The Z DNA appears by electron microscopy, to condense into toroidal structures (5, 2 6 ) . At low salt concentrations torolds do not necessarily form (27), however, at higher salt concentrations the toroids may aggregate further into banded trunk like structures (5). The self-aggregated Z DNA has been referred to as Z'DNA (25). It Is hypothesized that the left-handed helix, with its distinctive conformatlonal structure within a predominantly right-handed DNA matrix serves as a recognition signal for specific regulatory proteins. If Z DNA is recognized by specific proteins then the converse may also be true, where proteins that normally interact with B DNA would be restricted from normal protein - DNA interactions. We postulated such a possibility with the selected inhibition of the restriction enzyme Hae III hydrolyzing pBR 322 (28) at a site shown by Nordheim et.al. (22) to exist as Z DNA. Subsequent work from two laboratories (29, 30) with alternating GC sequences inserted into ccc DNA plasmlds appear to demonstrate that both a prokaryotic site specific DNA methyltransferase and a restriction endonuclease were relatively inactive at regions of Z DNA structures while fully active with B DNA sequences. Murlne and human DNA methyltransferases, however, did not show this differential activity with the two DNA forms (31). Vardlmon and Rich (29) and Zacharias and co-workers (30) have analyzed the interaction of site specific enzynes with ccc DNA containing both B and Z conformations. While these studies afford significant Insights Into proteln-DNA interactions the vast majority of eukaryotic DNA Is linear and may have conformational components not found In the circular molecule which may Influence these molecular Interactions. The studies in this report were conducted with linear DNA under conditions that were very different than 7238 a variety of biological functions including packaging DNA since Z DNA tends Nucleic Acids Research those employed with the circular DNA. The linear DNA was induced and maintained in the Z conformation by Co(NH3)6 3 + . The results we present here show that linear molecules of double-stranded poly (dGdC) in the Z Torn interact with site specific enzymes in a very different fashion than observed with Z-form GC sequences in the circular DNA molecules. The exact mechanism of the enzyme reaction with Z DNA cannot be resolved by current technologies. While antibody and physical methods can be employed to detect snail Z DNA regions within a B DNA molecule the interaction of site specific enzymes with Z DNA would effectively obscure regions of binding fron analysis of structure or shifts in the structural equilibrium. depends upon the procedure used to form the Z'DNA. The restriction endonucleases are completely Inactive with Z*DNA in 100 uN Co(NH3)6 3 + while soae residual activity was still detectable with the DNA •ethyltransferase enzyae. METHODS AND MATERIALS The polynucleotide poly(dG-dC) poly(dG-dC) lyophlllzed sodlua salt (Pharmacia P.L. Biochemlcals) was dissolved in distilled H2O at a concentration of 0.25 mg/al. It was dialyzed exhaustively against 25 mM Tris base, 1 mM EDTA, pH 7.5 before use (Robert F. Pasternack, personal communication). This DNA was diluted to 20 ug/al with a buffer consisting of 50 nH NaCl and 5 mM Trls-HCl at pH 8 prior to each subsequent study. The DNA was then shifted from B to Z and aggregated Z* form by the appropriate additions of 2 mM hexamalnecobalt (III) chloride (Ald (...truncated)