Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

Brahms, S.; Vergne, Jacques; Brahms, J.; Capua, E. Di; Bucher, Ph.; Koller, Th.

doi:10.1016/0022-2836(82)90539-3

Cited by 78 publications

(37 citation statements)

References 34 publications

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“…These results were previously reported by Brahms et al (12), and on that basis they assigned the polymer to the A conformatiop. We confirm their results.…”

Section: Methodssupporting

confidence: 80%

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Salt-induced Z-A-Z transition sequence in the mixed ribo-deoxyribo copolymer poly(rG-dC) X poly(rG-dC).

Wu¹,

Behe²

1984

Proc. Natl. Acad. Sci. U.S.A.

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The left-handed helical conformation of various polynucleotides has been seen in solution at higher salt concentration than has the right-handed helical conformation of the corresponding polymer. We report here, however, studies of conformational transitions in the mixed polymer poly-(rG-dC)-poly (rG-dC). This polynucleotide appears to exist in a Z conformation under low-salt conditions, converts to an A helix at intermediate salt, and reverts to a left-handed helix at high-salt concentration.The left-handed helical Z conformation (1) has been demonstrated to occur in the synthetic polydeoxynucleotide poly-(dG-dC)-poly(dG-dC) when the concentration of NaCl in solution is increased to >2.5 M (2). High concentrations of other mono-and oligovalent cations also can promote the transition (2, 3). Much smaller concentrations (<1 mM) of oligovalent ions are needed to cause the methylated analog of the above polymer, poly(dG-m5dC)-poly(dG-m5dC), to flip to the Z form (3). Other chemical modifications of poly-(dG-dC)poly(dG-dC), such as bromination (4) or alkylation (5-7), also can lower the critical concentration of cation necessary for the transition. However, in all of these examples, and also in the case of alternating purine-pyrimidine polymers containing A-T base pairs (8, 9), the Z conformation is the "high-salt" form-i.e., if the salt concentration is decreased, the conformational equilibrium is shifted toward the right-handed helical B form. An explanation that has been offered for this is that phosphate residues on different strands approach each other more closely in the Z form than in the B form, so that the Z conformation is favored by the electrostatic shielding afforded by higher salt concentrations (1). Another argument for the high-salt conformation being the Z form comes from the observation that Z DNA is less hydrated than B DNA (10), so that the Z conformation is favored by the lowered water activity of high-salt solutions.We report here the study of conformational transitions in the mixed ribo-deoxyribo copolymer poly(rG-dC)-poly(rGdC). We chose to study this polymer because ribonucleotides favor the C3'-endo conformation, and this sugar conformation was reported to occur in the guanosine residues of Z-DNA crystals (1). We have determined that, unlike previously studied polynucleotides, poly(rG-dC)-poly(rG-dC) appears to exist in a Z conformation at low-salt concentration (5 mM Tris), converts to the A conformation at intermediate levels of counterion, and again flips to a Z form at high NaCl concentration. MATERIALS AND METHODSSynthesis of the Polymer. Escherichia coli DNA polymerase I large fragment (Klenow fragment) was purchased either from Bethesda Research Laboratories or New England Biolabs. Poly(dI-dC)poly(dI-dC) was obtained from P-L Biochemicals, as were NTPs and dNTPs. Radioactive materials were purchased from ICN.The synthesis of the polymer was carried out as follows. The reaction medium contained in 1 ml 40 mM N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer (pH 8.95), 5 mM Mn C12...

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“…These results were previously reported by Brahms et al (12), and on that basis they assigned the polymer to the A conformatiop. We confirm their results.…”

Section: Methodssupporting

confidence: 80%

“…Brahms et al (12) have synthesized poly(rG-dC)-poly(rGdC) by a different method and have shown it to be in the A conformation at intermediate salt levels. We confirm this result (see below).…”

Section: Methodsmentioning

confidence: 99%

Salt-induced Z-A-Z transition sequence in the mixed ribo-deoxyribo copolymer poly(rG-dC) X poly(rG-dC).

Wu¹,

Behe²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…This was demonstrated by the study of intact plasmid DNAs and plasmid DNAs containing alternating d(pCpG) sequences (8)(9)(10)(11). Sequences in Z conformation were also found in form V DNA (12)(13)(14), form V being prepared from plasmid DNA by hybridizing covalently closed complementary single strands (15). These results also show that sequences other than alternating (dC-dG)n can be in Z conformation.…”

supporting

confidence: 57%

Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

Viegas‐Péquignot¹,

Derbin²,

Malfoy³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Antibodies against Z-DNA bind to fixed metaphase chromosomes of man and Cebus albifrons (Platyrrhini, Primate). By indirect immunofluorescence and indirect immunoperoxidase techniques, a heavy staining is detected in some segments of chromosomes of C. albifrons. These segments correspond to Rband-positive heterochromatin, which has a high G+C-base content. Euchromatin of human and Cebus chromosomes show a weak and heterogeneous staining that consistently reproduces an R-and T-banding pattern in both species. Because chromosome homologies previously were demonstrated between these distantly related species by chromosome banding, our results suggest that Z-DNA has been conserved during the course of primate evolution.Repeating sequences of alternating dC and dG deoxynucleotides can form the left-handed duplex DNA conformation, named Z-DNA, as demonstrated by x-ray diffraction studies of crystals of (dC-dG)n (1-3) and fibers of poly (dG-dC)-poly (dG-dC) ( (23, 24), the comparison of the staining patterns produced by antibodies to Z-DNA in euchromatin may be considered a check of the stability of Z-DNA conformation during chromosome evolution.In this work we show (i) that anti-Z-DNA antibodies bind to metaphase chromosomes of man and Cebus in a distinct and specific way and (ii) that an undisputable correspondence exists between the antibody binding and the chromosome banding patterns. In euchromatin, a weak but distinct and consistent staining is observed in the segments corresponding to the Rand T-bands, which are thought to be richer in G-C base pairs both in man and Cebus. Slight differences of intensity are observed from band to band: they are the same for all of the chromosomal segments of man and Cebus, which were found homologous by banding pattern and by gene mapping comparison (23)(24)(25). This fact underlines the stability of Z-DNA conformation during the evolution of mammalian chromosomes. Furthermore, the additional heterochromatic segments existing in Cebus chromosomes exhibit a consistent and very strong binding to anti-Z-DNA antibodies.MATERIAL AND METHODS Obtention of Metaphase Chromosomes. Metaphase cells were obtained from tissue cultures after skin biopsy of a female of C. albifrons held in captivity at the Museum National d'Histoire Naturelle, Paris.Metaphase cells from human lymphocytes were cultured according to usual methods (26). Cell cultures were treated by colchicine for 1 or 2 hr and then for 10 min with a hypotonic solution of diluted human serum (1:6, vol/vol). Cells were fixed in most of the experiments for at least 40 min with two different solutions (solution I, ethanol/chloroform/acetic acid, 6:3:1, vol/ vol; solution II, ethanol/acetic acid, 3:1, vol/vol). A prolonged fixation (45 min to 48 hr), with only the last fixative solution was used for some human cultures.The following banding methods were used to characterize the chromosomes of the specimen of C. albifrons: R-banding 5890 The publication costs of this article were defrayed in part by page charge payment. T...

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“…This site showed complete resistance to cleavage in form V DNA even at the end of 20 min (Fig 5, lane k). (-TWL) Single stranded melted regions, cruciform and left-handed helical structures, including Z-form will either contribute to zero or negative twist, therefore, It has been shown earlier that 35-40% of the sequences in form V molecule indeed adopt non-B conformation including Zform (16,20). Eventually, writhe in form V molecule is decided by the potential of sequences to adopt left-handed helical conformations and other hydrogen bonded structures.…”

Section: Resultsmentioning

confidence: 99%

“…10 units of enzyme was added and the reaction was carried out at 370C. 20 Id aliquots were taken at different time intervals and the reaction was arrested by the addition of stop mix. For single hit analysis, 0.1 ,ug of form I or form V DNA was digested in 50 ,ul of medium salt reaction buffer.…”

Section: Methodsmentioning

confidence: 99%

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe

Shouche

Ramesh

Brahachari

1990

Nucleic Acids Research

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The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHl, Pstl, Aval and HindlIl could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as Narl and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences. INTRODUCTIONOver the last decade our understanding of DNA structure as a rigid uniform double helix has significantly changed to that of a sequence-dependent dynamic conformation (1,2). Although several crystal structures of oligonucleotides have shown that the DNA conformation is indeed sequence-dependent (3), paucity of data does not enable us to arrive at a generalised rule. However, the influence of ionic environment and topological strain (resulting from supercoiling) in altering DNA secondary structure has provided valuable information with regard to structural transition from B to Z form (4-8). Enzymes such as DNase I and SI nuclease have been extensively used to probe DNA conformation in solution (2,9-11). Although restriction endonucleases are believed to be only sequence-specific and not structure sensitive, recent studies have shown that some restriction enzymes fail to recognize and cleave DNA in left handed Z conformation (12)(13)(14).

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Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

Cited by 78 publications

References 34 publications

Salt-induced Z-A-Z transition sequence in the mixed ribo-deoxyribo copolymer poly(rG-dC) X poly(rG-dC).

Salt-induced Z-A-Z transition sequence in the mixed ribo-deoxyribo copolymer poly(rG-dC) X poly(rG-dC).

Z-DNA immunoreactivity in fixed metaphase chromosomes of primates.

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe

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