Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

AT base pairs are less stable than GC base pairs in Z-DNA: The crystal structure of d(m5CGTAm5CG)

Two hexanucleoside pentaphosphates, 5-methyl and 5-bromo cytosine derivatives of d(CpGpTp-ApCpG) have been synthesized, crystallized, and their three-dimensional structure solved. They both form left-handed Z-DNA and the methylated derivative has been refined to 1.2 Å resolution. These are the first crystal Z-DNA structures that contain AT base pairs. The overall form of the molecule is very similar to that of the unmethylated or the fully methylated (dC-dG)₃ hexamer although there are slight changes in base stacking. However, significant differences are found in the hydration of the helical groove. When GC base pairs are present, the helical groove is systematically filled with two water molecules per base pair hydrogen bonded to the bases. Both of these water molecules are not seen in the electron density map in the segments of the helix containing AT base pairs, probably because of solvent disorder. This could be one of the features that makes AT base pairs form Z-DNA less readily than GC base pairs.     

Methylation of cytosine in the 5-position alters the structural and energetic properties of the supercoil-induced Z-helix and of B-Z junctions

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence specificity of streptozotocin-induced mutations.

The isolation and characterization of streptozotocin (STZ)-induced mutations in the phage P22 mnt repressor gene is described. Cells carrying the plasmid-borne mnt gene were exposed to STZ to give 10-20 percent survival and at least an eleven-fold increase in mutation frequency. DNA sequence analysis showed that 50 of 51 STZ-induced mutations were GC to AT transitions, and one was an AT to GC transition. We have also compared the STZ mutational spectrum to that for N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). There are sites in the mnt gene which are mutated only by STZ; only by MNNG, or by both agents. Sites at which only STZ induced GC to AT transition mutations occur were in sequences that are pyrimidine rich 5' to the mutated site and purine rich 3' to the mutated site. Induction of mutations by both STZ and MNNG should be considered to maximize the number of mutable sites.     

Parallel-stranded DNA with natural base sequences

Noncanonical parallel-stranded DNA double helices (ps-DNA) comprising natural nucleotide sequences are usually second in stability to antiparallel-stranded (aps) canonical DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA with mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of Watson-Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into the both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylcytosine were shown to form ps-DNA with transAT-, trans isoGC, and trans iso5meCG pairs exceeding in stability a related aps duplex. (3) Nucleotide sequence dG(GT)4G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT)n and d(GnTm) sequences able to form quadruplexes, the dG(GT)4G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT)n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed.     

Formation of DNA triple helices incorporating blocks of G.GC and T.AT triplets using short acridine-linked oligonucleotides.

We have used DNase I footprinting to assess triple helix formation at target sites containing the sequences A6G6.C6T6 and G6A6.T6C6. These sequences can be recognized by the acridine-linked oligopyrimidines Acr-T5C5 and Acr-C5T5 respectively at low pH, using well-characterised T.AT and C+.GC triplets. At pH 7.5 A6G6.C6T6 is specifically bound by Acr-G5T5, utilising G.GC and T.AT triplets in which the third strand runs antiparallel to the purine strand of the duplex. This interaction requires the presence of magnesium ions. No interaction was detected with Acr-T5G5, an oligonucleotide designed to form parallel G.GC and T.AT triplets. In contrast neither Acr-T5G5 nor Acr-G5T5 produced DNase I footprints with the target sequence G6A6.T6C6. These results suggest that, in an antiparallel R.RY triple helix, the T.AT triplet is weaker than the G.GC triplet. We find no evidence for the formation of structures containing parallel G.GC triplets.     

Transition of a cloned d(AT)n-d(AT)n tract to a cruciform in vivo.

A 34 base pair tract of the simple repeating dinucleotide d(AT)n-d(AT)n cloned into a 2.4 kb polylinker plasmid vector undergoes a structural transition in response to negative superhelical coiling. The transition has been characterized by 2 dimensional gel electrophoresis, mapping of S1, P1 and T7 endonuclease 1 sensitive sites, and mapping of sites that are sensitive to modification by bromoacetaldehyde. After S1 nuclease treatment it is possible to trap supercoiled species that are nicked on one or both strands near the center of the palindrome. These data show that the alternate state adopted by the d(AT)n-dAT)n insert is a cruciform rather than a Z conformation. Unlike other B-cruciform transitions the transition in d(AT)n-d(AT)n has a low activation energy and the transition is facilitated by the presence of magnesium ions. Evidence from in-vivo topoisomer distributions is presented which shows that under conditions of blocked protein synthesis the d(AT)n-d(AT)n insert will spontaneously adopt the cruciform state in-vivo in E. coli.     

Long-range structural effects in supercoiled DNA: statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817-827] a set of sequences, called C-type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C-type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S-type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations: 1. the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C-type character; 2. C-type induction by an A + T rich Drosophila sequence; 3. low-temperature cruciform extrusion by an (AT)34 sequence; 4. the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat; 5. the effects of systematic deletion of the colL sequence; and 6. the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat. These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.     

Parallel-Stranded DNA with Natural Base Sequences

Noncanonical parallel-stranded DNA double helices (ps-DNA) of natural nucleotide sequences are usually less stable than the canonical antiparallel-stranded DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in regions of trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA of mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of a Watson–Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylisocytosine were shown to form ps-DNA with transAT-, trans isoGC, and transiso^5meCG pairs exceeding in stability a related canonical duplex. (3) Nucleotide sequence dG(GT)₄G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT) _n - and d(G _n T _m ) sequences able to form quadruplexes, the dG(GT)₄G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT) _n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed.     

The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study.

The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.     

In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is discussed.     

A structural transition in d(AT)n.d(AT)n inserts within superhelical DNA

We have constructed plasmids carrying d(AT)n.d(AT)n inserts of different lengths. Two-dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease S1, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)n.d(AT)n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 A resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg(2+) at 1.96 A resolution. The protein structure consists of an N-terminal domain with an alpha/beta-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed beta-sheet helix (LbetaH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed beta-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed beta-sheet helix domains. UDP-N-acetylglucosamine and Mg(2+) are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg(2+) and the side-chain of Lys22.     

Intermediate range effects in DNA. I: Low pH/stress induced conformational changes in the vicinity of an extruded d(AT)n.d(AT) in cruciform.

A family of plasmids which contain d(AT)n cruciforms are sensitive to "single strand specific" (SS) endonucleases and a variety of chemical probes in a "random sequence" region centered 10-30 residues away from the cruciform junction. The SS nuclease sensitive structures are dependent on the presence of the extruded cruciform and exhibit a degree of sequence independence. Their appearance depends upon the combined effects of slightly lower than neutral pH and superhelical coiling above the minimum required to drive the extrusion of the d(AT)n cruciform arms. The nuclease sensitive structure is therefore underwound with respect to the B conformation and contains protonated bases.     

Calculation of melting curves for DNA

A method is reported for calculating the melting curve of a DNA molecule of random base sequence, including in the formalism the dependence of the free energy of base pair formation on the size of a denatured section. Some explicit results are shown for a “typical” base sequence, in particular the probability of helix formation at individual base pairs in several different regions of the molecule and the amount of melting from the end of the chain. Particular attention is drawn to the variation of local melting behavior from one region of the molecule to another. It is found that sections rich in AT melt at relatively low temperatures with a fairly broad transition curve, whereas regions rich in GC pairs melt at higher temperatures (as expected) with a very abrupt, local transition curve. To account qualitatively for the results one may divide melting into two kinds of processes: (a) the nucleation and growth of denatured regions, and (b) the merging together of two denatured sections at the expense of the intervening helix. The first of these processes dominates in the first stages of melting, and leads to rather broad local melting curves, whereas the second process predominates in the later stages, and occurs, in a particular part of the molecule, over a very narrow temperature range. It is estimated that the average length of a helix plus adjacent coil section at the midpoint of the transition is approximately 600 base pairs. Since transition curves which measure the local melting behavior reflect local compositions fluctuations, these curves contain information about the broad outlines of base sequence in the molecule. Some suggestions are made concerning experiments by which this potential information source could be exploited. In particular, it is pointed out that one might hope to map AT or GC rich regions at particular genetic loci in a biologically active DNA molecule. Values of the relevant parameters found earlier for the transition of homopolymers produce melting curves for a DNA of random base sequence which are in good agreement with the experimental transition curve for T2 phage DNA. Hence the present theoretical picture of the melting of polynucleotides is at least internally self-consistent.     

Characteristics of Z-DNA helices formed by imperfect (purine-pyrimidine) sequences in plasmids

The capacities of three synthetic sequences to adopt left-handed helices were evaluated in recombinant plasmids. The sequences consisted of very short runs of (CG)n (n = 2-4) interspersed with runs of alternating A.T base pairs and/or with regions of non-alternating base pairs. The plasmids were studied by two-dimensional gel electrophoresis to determine the natures of the conformational transitions and their free energies of formation. These results coupled with analyses with chemical (diethyl pyrocarbonate, osmium tetroxide, and bromoacetaldehyde) and enzymatic (S1 nuclease, T7 gene 3 product, and MHhaI) probes indicated that the entire sequence was adopting a left-handed helix in each case. In one of these sequences, Z-DNA formation necessitated the retention of the anti conformation of one of the guanines in a region of non-alternation. In a sequence which contains out-of-phase regions of alternation, our results indicate the formation of a separate left-handed helix in the central (CG)2 region, thus forming two Z-Z junctions. In summary, we conclude that only very short regions of alternating CG are necessary to effect the B to Z transition and that this conformational change can be transmitted through non-alternating regions. A set of empirical rules governing the characteristics of the B to Z transition and the types of left-handed helices in supercoiled plasmids was derived from studies on a systematic series of 17 plasmids.     

DNA polymorphism: spectroscopic and electro-optic characterizations of Z-DNA and other types of left-handed helical structures induced by Ni2+

CD and uv spectroscopy reveal that the synthetic polynucleotides poly(dG–dC) · poly(dG–dC) and poly(dG–m⁵dC) · poly(dG–m⁵dC) undergo a transition induced by small amounts of Ni⁺⁺ cation from a right-handed B-form to left-handed Z-type conformations. We describe the application of steady-state and transient electric birefringence to the characterization of the transition observed at very low ionic strength (10 mM Tris HCl, pH 7.4). Dialysis experiments show that the changes in spectroscopic and electro-optic properties upon addition of Ni⁺⁺ are completely reversible. The differences in shape of the inverted CD spectra suggest the existence of a family of left-handed conformations, depending on the polymer used and on the amounts of cation added. The stoichiometry required for inducing the Z-conformation of poly(dG–m⁵dC) is 1 cation/4 nucleotide phosphates. The transition is accompanied by a decrease in birefringence, an increase in length, and the more important contribution of a permanent or slowly induced dipole moment to the orientation mechanism. High concentrations of Ni⁺⁺ promote the Z → Z* transition. Upon increasing the Ni⁺⁺ concentration, poly(dG–dC) undergoes a biphasic transition, first to one intermediate conformation that is neither B- nor Z-like and then to a left-handed form that is probably different from Z*. These conversions are accompanied by regular decreases both in birefringence and in chain length, but no transient appears in the field-reversal experiments.