Extraordinary stable structure of short single-stranded DNA fragments containing a specific base sequence: d(GCGAAAGC).

Short single-stranded DNA fragments carrying a GCGAAAGC sequence were found to move unexpectedly faster than other fragments of the same length in electrophoresis on a polyacrylamide gel containing a denaturing agent. The fragments were noted to have a stable structure even in 7M urea solution, but the stability cannot be explained simply on the basis of base pair formation alone. Physical characterization of the GCGAAAGC fragment indicated that it takes a hairpin-like structure in spite of the short chain length with only two G-C base pairs, comprised of GCG and AAAGC subsegments, each possessing a helical configuration independent of the others. Some biological implications of this unusual structure are discussed.     

DNA octaplex formation with an I-motif of water-mediated A-quartets: reinterpretation of the crystal structure of d(GCGAAAGC)

The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and reasonably refined including the disordered residues. The two DNA strands form a base-intercalated duplex, and the four duplexes are assembled according to the crystallographic 222 symmetry to form an octaplex. In the central region, the eight strands are associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium cations link the four duplexes to support the octaplex formation. Based on these structural features, a proposal that folding of d(GAAA)n, found in the non-coding region of genomes, into an octaplex can induce slippage during replication to facilitate length polymorphism is presented.     

CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase.

CircumVent thermal cycle and standard DNA sequencing protocols utilizing the cloned and highly thermostable VentR (exo-) DNA polymerase are described. The thermal cycle sequencing procedures are advantageous because they allow fast and simple semiautomation of the sequencing reaction; make possible the direct DNA sequencing of PCR products, bacterial colonies and phage plaques; require only femtomoles of template DNA; eliminate the requirement of an independent primer annealing step; remove the requirement of denatured plasmids for sequencing double-stranded templates; and use a highly thermostable DNA polymerase for sequencing through potential recalcitrant secondary structure domains and large linear double-stranded DNA templates such as lambda derivatives. More standard methods of DNA sequencing (i.e., a one-step protocol and a labeling-termination protocol) are also presented. For each protocol, alternatives for choice of label and method of labeling are presented, including the use of 5' biotinylated primers for chemiluminescent DNA sequencing and fluorinated primers for automated sequencing using the BaseStation Automated DNA Sequencer.     

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.     

Sticky DNA: effect of the polypurine.polypyrimidine sequence

The polypurine.polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA.TC)(37), which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA.TCC)(n) inserts (where n = 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA.TTC)(150) (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA.TCCTTC)(65) of the same approximate length (390 bp). A sequence that is replete with polypurine.polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA.TTC)(n) (where n = 176 or 80) readily formed sticky DNA with (GAAGGA.TCCTTC)(65) cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.     

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.     

Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT)

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.     

N.m.r. and c.d. studies of the DNA fragments d(TATATATA) and d(TATATA) in solution

DNA fragments d(TATATATA) and d(TATATA) were studied in low-salt aqueous solutions and found to coexist in more than one conformer. 1H-n.m.r. demonstrates that single-stranded and double-stranded states are involved in the conformational coexistence. Circular dichroism spectroscopy indicates a global B-DNA stacking of bases in the fragments. 31P-n.m.r. resonances of the TpA and ApT phosphodiester bonds are substantially separated in the spectra of both d(TATATATA) and d(TATATA) duplexes to suggest an alternating architecture of their backbones. In fact, the oligonucleotide duplexes are much more alternating than the corresponding polynucleotide under the same solution conditions. The alternating character of the d(TATATATA) double helix is further enhanced in molar caesium fluoride solutions. The oligonucleotide isomerization into X-DNA is, however, accompanied by gel formation, which makes high resolution n.m.r. measurements impossible.     

Energetics of the B-H transition in supercoiled DNA carrying d(CT)x.d(AG)x and d(C)n.d(G)n inserts.

We have studied the B-H transition in the d(AG)x inserts of varying length under superhelical stress. The new data and previously published results for the d(G)31 insert are treated within a phenomenological model of the B-H transition, making it possible to obtain, for the first time, the energy parameters of the B-H transition in the d(AG)x and d(G)n sequences.     

Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH₂ functionalities. The PBD dimer SJG-136, which has a C8–O–(CH₂)₃–O–C8′′ central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8–O–(CH₂)₅–O–C8′), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8′-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA–interaction behaviour of a PBD dimer of particular C8–C8′ linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.     

Conformational properties and thermodynamics of the RNA duplex r(CGCAAAUUUGCG)2: comparison with the DNA analogue d(CGCAAATTTGCG)2.

The thermodynamic stability of nine dodecamers (four DNA and five RNA) of the same base composition has been compared by UV-melting. TheDeltaG of stabilisation were in the order: r(GACUGAUCAGUC)2>r(CGCAAATTTGCG)2 approximately r(CGCAUAUAUGCG)2>d(CGCAAATTTGCG)2 approximately r(CGCAAAUUUGCG)2>d(CGCATATATGCG)2 approximately d(GACTGATCAGTC)2>r(CGCUUUAAAGCG)2 approximately d(CGCTTTAAAGCG)2. Compared with the mixed sequences, both r(AAAUUU) and r(UUUAAA) are greatly destablising in RNA, whereas in DNA, d(TTTAAA) is destabilising but d(AAATTT) is stabilising, which has been attributed to the formation of a special B'structure involving large propeller twists of the A-T base pairs. The solution structure of the RNA dodecamer r(CGCAAAUUUGCG)2has been determined using NMR and restrained molecular dynamics calculations to assess the conformational reasons for its stability in comparison with d(CGCAAATTTGCG)2. The structures refined to a mean pairwise r.m.s.d. of 0.89+/-0.29 A. The nucleotide conformations are typical of the A family of structures. However, although the helix axis displacement is approximately 4.6 A into the major groove, the rise (3.0 A) and base inclination ( approximately 6 degrees ) are different from standard A form RNA. The extensive base-stacking found in the AAATTT tract of the DNA homologue that is largely responsible for the higher thermodynamic stability of the DNA duplex is reduced in the RNA structure, which may account for its low relative stability.     

Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg),d(cCNNGg), and d(gCNNGc)

Hairpins play important roles in the function of DNA, forming cruciforms and affecting processes such as replication and recombination. Temperature gradient gel electrophoresis (TGGE) and in vitro selection have been used to isolate thermodynamically stable DNA hairpins from a six-nucleotide random library. The TGGE-selection process was optimized such that known stable DNA tetraloops were recovered, and the selection appears to be exhaustive. In the selection, four families of exceptionally stable DNA loops were identified: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc). (Lowercase denotes the closing base pair; N = A, C, G, or T; and B = C, G, or T.) It appears that the known stable d(cGNAg) triloop motif can be embedded into a tetraloop, with the extra nucleotide inserted into either the middle of the loop, d(cGNNAg), or at the 3'-end of the loop, d(cGNABg). For d(cGNNAg) and d(cGNABg), a CG closing base pair was strongly preferred over a GC, with DeltaDeltaG degrees (37) approximately 2 kcal/mol. Members of the two families, d(cCNNGg) and d(gCNNGc), are similar in stability. The loop sequences and closing base pairs identified for exceptionally stable DNA tetraloops show many similarities to those known for exceptionally stable RNA tetraloops. These data provide an expanded set of thermodynamic rules for the formation of tetraloops in DNA.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.     

Nuclear proteins that bind the pre-mRNA 3'' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.     

Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA.

A protein which selectively binds d(GA)n and d(GT)n sequence repeats in single stranded DNA has been identified in human fibroblasts. This protein, designated PGB, has been purified at least 500-fold by ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography and affinity chromatography in a column of d(GA)-Sepharose. Electrophoretic mobility shift assays revealed that the PGB protein bound most avidly d(GA)n and d(GT)n tracts of n > 5. It also bound other G-rich DNA sequence repeats, including dGn tracts, with lower affinities. It did not manifest significant binding affinities to single stranded M13 DNA, or to the homopolynucleotides poly dA, poly dC and poly dT, or to various DNA sequence repeats which do not contain G residues, such as d(A-C)n and d(TC)n. It did not bind double stranded d(T-C)n.d(GA)n tracts or other double stranded DNA sequences. In glycerol gradient centrifugation assays the d(GA)n- and the d(GT)n-binding activities cosedimented as a homogeneous protein species having an S20,w = 9.4 +/- 0.7 and an estimated native molecular weight of 190,000 +/- 7,000. UV crosslinking assays revealed that the protein contains 33.6 +/- 2.1 kd subunits which bind d(GA)n and d(GT)n sequences. However, SDS-polyacrylamide gel electrophoresis of the purified protein followed by silver staining indicated that it may also contain other subunits that do not contact the DNA. It is proposed that binding of the PGB protein to single stranded d(GA)n or d(GT)n tracts in double stranded topologically restricted DNA may stimulate strand separation and formation of triple helices or other unusual DNA structures.     

DNA methylase: purification from ascites cells and the effect of various DNA substrates on its activity.

DNA methylase has been purified 405-fold from Krebs II ascites cells. The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine. Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations. Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it. By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them. Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA. DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.     

The effect of Cr(III) upon the thermal denaturation of DNA

Thermal denaturation studies of Cr-DNA solutions at pH 6.0 were carried out by monitoring the uv absorbance at 260 nm. The melting curves of solutions of calf thymus and Escherichia coli DNA with added Cr(ClO₄)₃ were broadened and shifted to higher temperatures. As the ratio of Cr: DNA increased, the melting temperature increased until it reached a maximum at Cr: DNA ratios of 0.7 (E. coli) and 0.9 (calf thymus). At higher concentrations of Cr³⁺ the melting temperature decreased and then leveled off, but it never fell as low as that of the pure DNA.