Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine alternating sequences which are known to adopt the left-handed Z-DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z-motifs in the SV40 minichromosome. We have analyzed the presence of Z-DNA through the change in linkage which should accompany formation of this left-handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z-DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right-handed B-DNA conformation in vivo.     

Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.     

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.     

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.     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

Sequences that adopt non-B-DNA conformation in form V DNA as probed by enzymic methylation

pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero. We have used sequence-specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA. Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation. Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences. Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure. In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA. These non-B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain. Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed. In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.     

Nonalternating purine pyrimidine sequences can form stable left-handed DNA duplex by strong topological constraint

In vivo, left-handed DNA duplex (usually refers to Z-DNA) is mainly formed in the region of DNA with alternating purine pyrimidine (APP) sequence and plays significant biological roles. It is well known that d(CG)_n sequence can form Z-DNA most easily under negative supercoil conditions, but its essence has not been well clarified. The study on sequence dependence of Z-DNA stability is very difficult without modification or inducers. Here, by the strong topological constraint caused by hybridization of two complementary short circular ssDNAs, left-handed duplex part was generated for various sequences, and their characteristics were investigated by using gel-shift after binding to specific proteins, CD and T_m analysis, and restriction enzyme cleavage. Under the strong topological constraint, non-APP sequences can also form left-handed DNA duplex as stable as that of APP sequences. As compared with non-APP sequences, the thermal stability difference for APP sequences between Z-form and B-form is smaller, which may be the reason that Z-DNA forms preferentially for APP ones. This result can help us to understand why nature selected APP sequences to regulate gene expression by transient Z-DNA formation, as well as why polymer with chirality can usually form both duplexes with left- or right-handed helix.     

Z-DNA的形成及其与基因转录的关系

Z-DNA是一种非常独特的DNA二级结构.与B-DNA相比,Z-DNA最显著的结构特征是左手螺旋和磷酸-核糖骨架呈“zigzag”状. 虽然目前对Z-DNA功能的了解还不确切,但毫无疑问,Z-DNA与基因的转录和调控密切相关. 一方面,在体内Z-DNA在基因转录过程中产生;另一方面,分布于启动子等不同区域的Z-DNA又可以反过来调控基因的转录, 即Z-DNA能够增强一些基因转录,也能抑制某些基因的表达,但其调控机制还不清楚.这种调控似乎与Z-DNA在启动子中的位置、基因和细胞类型有关.研究Z-DNA的形成及其与基因转录的关系对理解基因转录调控理论具有十分重要的意义.     

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences.

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.     

Non-B DNA conformations, mutagenesis and disease

Recent discoveries have revealed that simple repeating DNA sequences, which are known to adopt non-B DNA conformations (such as triplexes, cruciforms, slipped structures, left-handed Z-DNA and tetraplexes), are mutagenic. The mutagenesis is due to the non-B DNA conformation rather than to the DNA sequence per se in the orthodox right-handed Watson-Crick B-form. The human genetic consequences of these non-B structures are approximately 20 neurological diseases, approximately 50 genomic disorders (caused by gross deletions, inversions, duplications and translocations), and several psychiatric diseases involving polymorphisms in simple repeating sequences. Thus, the convergence of biochemical, genetic and genomic studies has demonstrated a new paradigm implicating the non-B DNA conformations as the mutagenesis specificity determinants, not the sequences as such.     

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.     

The rare crystallographic structure of d(CGCGCG)(2): the natural spermidine molecule bound to the minor groove of left-handed Z-DNA d(CGCGCG)(2) at 10 degrees C

Several crystal structure analyses of complexes of synthetic polyamine compounds, including N(1)-(2-(2-aminoethylamino))ethyl)ethane-1,2-diamine PA(222) and N(1)-(2-(2-(2-aminoethylamino)ethylamino)ethyl)ethane-1,2-diamine PA(2222), and left-handed Z-DNA d(CGCGCG)(2) have been reported. However, until now, there have been no examples of naturally occurring polyamines bound to the minor groove of the left-handed Z-DNA of d(CGCGCG)(2) molecule. We have found that spermidine, a natural polyamine, is connected to the minor groove of left-handed Z-DNA of d(CGCGCG)(2) molecule in a crystalline complex grown at 10 degrees C. The electron density of the DNA molecule was clear enough to determine that the spermidine was connected in the minor groove of two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2). This is the first example that a spermidine molecule can form a bridge conformation between two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2) in the minor groove.     

Left-handed Z-DNA

Since the Watson-Crick proposal of right-handed B-DNA, numerous studies have been devoted to the conformation of DNA. Both natural DNAs of heterogeneous sequences and synthetic DNAs are capable of adopting more than one conformation. The specific conformation a DNA adopts appears to depend mainly on its base sequence and its environmental conditions. For a given DNA, changes in environmental conditions can induce conformational transitions which occur according to cooperative or non-cooperative processes (for general reviews see Ref. 1a, b). Despite many results, molecular biologists did not put much emphasis on the polymorphism of DNA. The discovery of the intraconversion in helical sense between the right-handed B and left-handed Z conformers of DNA has brought a new interest in the polymorphism of DNA. It is now proposed that this polymorphism has important functions in biological reactions. A recent review, ‘The Chemistry and Biology of Left-handed Z-DNA’, by Rich et al. [2] has just been published. We here report some of the results published in 1984 on Z-DNA.     

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.     

Hybrid hexanucleotide duplex containing cyclonucleotides and deoxynucleotides: the d(TA) segment can adopt a high anti left-handed double-helical structure

It is known that oligonucleotides containing cyclonucleosides with a high anti (intermediate between anti and syn) glycosidic conformation adopt left-handed, single- and double-helical structures [Uesugi, S., Yano, J., Yano, E., & Ikehara, M. (1977) J. Am. Chem. Soc. 99, 2313-2323]. In order to see whether DNA can adopt the high anti left-handed double-helical structure or not, a self-complementary hexanucleotide containing 6,2'-O-cyclocytidine (C(o)), 8,2'-O-cycloguanosine (G(o)), thymidine, and deoxyadenosine, C(o)G(o)dTdAC(o)G(o), was synthesized. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C(o)G(o)dTdAC(o)G(o) adopts a left-handed double-helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. A conformational model of the left-handed duplex was obtained by calculation with energy minimization. Thus it appears that DNA can form a high anti, left-handed double helix under some constrained conditions, which is quite different from that of Z-DNA.     

Left-handed Z-DNA binding by the recA protein of Escherichia coli

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.     

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me⁵dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent Z ⇌ B transition it has been shown that a minor fluctuation in Na⁺ concentration at ambient temperature can bring about B to Z transition. Forthe first time, wehave observed a novel Z⇌B⇌Zuble transition in poly(dG-Me⁵dC) as the Na⁺ concentration is gradually increased. This suggests that a minor fluctuation in Na⁺ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pβG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (L _k = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pβG and pβR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.