Structure of (dG)n · (dC)n Under Superhelical Stress and Acid pH

Abstract

We have recently shown that a (GA)_n · (TC)_n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 327 (1985)). Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)_n · (dC)_n insert to undergo a pH-dependent structural transition similar to that of the (GA)_n · (TC)_n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3,667 (1986)).     

Raman Spectroscopy Study of the B-Z Transition in (dG-dC)n · (dG-dC)n and a DNA Restriction Fragment

Abstract

The B to Z conformational transition of (dG-dC)_n·(dG-dC)_n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831–833 and 1093 cm^?1. Both (dG-dC)_n·(dG-dC)_n and the 157 bp DNA show changes in the guanine vibration at 682 cm^?1 and backbone band at 831–3 cm^?1 preceeding a highly cooperative change in the 1093 cm^?1 PO 7 vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway.

We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)_n·(dG- dC)_n indicate that in 4.5 M NaC/the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.     

Abundance and degree of dispersion of genomic d(GA) n ·d(TC) n sequences

Summary The abundance of d(GA) _n ·d(TC) _n tracts was determined in genomes of rodents and primates. Dot blot hybridization assays revealed that such tracts constitute 0.40%, 0.30%, and 0.40%, respectively, of the rat, hamster, and mouse genomes, but only 0.07% and 0.05% of the human and monkey genomes. A plaque hybridization assay of rat and human genomic libraries showed that 37% and 16%, respectively, of the recombinant phages in these libraries contain d(GA) _n ·d(TC) _n tracts. A survey of sequences stored in the GenBank data bank showed that a significant fraction of the stored rodent genes (about 2.0%) contain long d(GA) _n ·d(TC) _n tracts (n> 30) with <10% mismatching. The primate genes contain only shorter tracts (n<15) with <10% mismatching. In addition, the rodent and the primate genes contain tracts with larger degrees of mismatching. The chicken, which represents an entirely different branch of the evolutionary tree, was found to be as low in d(GA) _n ·d(TC) _n tracts as the primates. It is suggested that a common ancestor of the rodents has acquired the ability to amplify d(GA) _n ·d(TC) _n tracts.     

Parallel-Stranded Oligonucleotides with Alternating d(A-isoG)/d(T·C) and d(A·G)/d(T·m5isoC) Sequences

Abstract

Parallel-stranded (ps) DNA hairpins with alternating d(A-isoG)/d(T·C) (designated as ps-t1) and d(A·G)/d(T·m⁵isoC) (ps-t2) sequences were studied by means of UV, CD and fluorescence spectroscopy. The thermostability of d(A·G)/d(T·m⁵isoC) sequence was close to that of aps d(G·A)/d(T·C). The stability of the ps d(A·isoG)/d(T·C) sequence was even higher than that of a related anti-parallel-stranded (aps) d(G·A)/d(T·C) sequence, being unique for ps DNAs studied so far.     

Laser Photofootprinting of d(C)·d(G)·d(G) Intramolecular Triplex

Abstract

Supercoiling-induced structural transition of the d(C₂₄GC₂₁,) · d(G₂₁CG₂₄) sequence in plasmid DNA in the presence of Mg²⁺ at neutral pH results in alterations of efficiencies of not only single-quantum (pyrimidine[6–4]pyrimidone adducts) but also two-quantum (alkalisensitive lesions of dG residues) photomodifications of nucleoside residues within this sequence. The generation of both types of photoreactions was achieved by the application of high-intensity laser UV radiation (intensity ～ 10¹¹ W/m², pulse duration ～ 10^?8 s, λ= 266 nm) for irradiation of a plasmid DNA The modification extent sufficient for analysis of photoreaction efficiency distributions along both strands of the insert (photofootprinting) was obtained by the action of a single nanosecond pulse of laser UV radiation. The pattern of a laser photofootprinting is consistent with the d(C) · d(G) · d(G) triplex formation in the presence of Mg²⁺ within the insert and shows some details of this triplex structure.     

Preparation and separation of d(pT)10·n oligonucleotides

A series of oligomers having the general formula d(pT)_10·n, n varying from 2 to 20, has been prepared by enzymatic joining of d(pT)₁₀, annealed on poly dA, employing T₄ polynucleotide ligase. The oligomers could be separated on 8 or 12% polyacrylamide gels. Such oligomers may prove useful as molecular weight markers and as initiators for various polymerases.     

Interpretation of DNA vibration modes: I-The guanosine and cytidine residues involved in poly(dG-dC)·poly(dG-dC) and d(CG)3·d(CG)3

Abstract

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5′-dGMP and 2′-deoxycytidine molecules to be reproduced. The role of both C2′-endo and C3′-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (χ), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm^?1 and 785-500 cm^?1 regions, when the right → left-handed conformational transition occurs.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Interferon inducing activity of (A)n·(U)n complexes of varying chain length

Evidence is presented that the interferon-inducing activity of (A)_n·(U)_n in primary rabbit kidney cells with respect to the chain length of the constituting (A)_n and (U)_n strands is governed by the following criteria: (1) the activity increases with the length of the uninterrupted double-stranded segment in the complex whereby both chains are equally important and the number of such segments for complex molecule is without effect, (2) at a constant total concentration of constituting nucleotides, the activity increases with the number of double-stranded molecular entities available to the cell, and (3) complexes with the (U)_n strand considerably overlapping the (A)_n strand are inactive due to the formation of triple-stranded structures.     

Urbanicity and Paediatric Bacteraemia in Ghana—A Case-Control Study within a Rural-Urban Transition Zone

Background

Systemic bacterial infections are a major cause of paediatric febrile illness in sub-Saharan Africa. Aim of this study was to assess the effects of social and geographical determinants on the risk of bacteraemia in a rural-urban transition zone in Ghana.

Methods

Children below 15 years of age with fever were recruited at an outpatient department in the suburban belt of Kumasi, Ghana’s second largest city. Blood was taken for bacterial culture and malaria diagnostics. The socio-economic status of participants was calculated using Principle Component Analysis. A scale, based on key urban characteristics, was established to quantify urbanicity for all communities in the hospital catchment area. A case-control analysis was conducted, where children with and without bacteraemia were cases and controls, respectively.

Results

Bacteraemia was detected in 72 (3.1%) of 2,306 hospital visits. Non-typhoidal Salmonella (NTS; n = 24; 33.3%) and Salmonella typhi (n = 18; 25.0%) were the most common isolates. Logistic regression analysis showed that bacteraemia was negatively associated with urbanicity (odds ratio [OR] = 0.8; 95% confidence interval [CI]: 0.7–1.0) and socio-economic status (OR = 0.8; 95% CI: 0.6–0.9). Both associations were stronger if only NTS infections were used as cases (OR = 0.5; 95% CI: 0.3–0.8 and OR = 0.6; 95% CI: 0.4–1.0, respectively).

Conclusions

The results of this study highlight the importance of individual as well as community factors as independent risk factors for invasive bacterial infection (IBI) and especially NTS. Epidemiological data support physicians, public health experts and policy makers to identify disease prevention and treatment needs in order to secure public health in the transitional societies of developing countries.     

溶液中d(AT)_6的构象转化

 本文报道了溶液中d(AT)_6构象研究的结果。探讨了d(AT)_6的物化特征。d(AT)_6在0.05mol/L NaCl和4.5 mol/L NaCl中的UV和CD谱是典型的Z-和B-型DNA谱,UV熔化曲线形状依赖于盐的浓度。当盐浓度低于0.05 mol/L NaCl时,曲线有负斜率;而盐浓度大于0.05mol/L NaCl时,曲线的斜率变为正,即随盐浓度增加,T_m值增加。在链浓度的依赖性基础上,计算了两种构象的△H和△S。     

Flexibility of A · Pairs in Left-Handed DNA Helices

Abstract

We have analysed by various approaches the structure of cloned synthetic sequences in supercoiled plasmids. Individual inserts were formed by d(C-G)_n blocks interrupted by the presence of A · T pairs positioned either in phase or out of phase of pur-pyr alternation. Based on the thermodynamic analysis we obtained results confirming that A · T pairs are easily incorporated into left-handed helices without significant energetic penalty. Sequences GTAC which are known to form cruciform structures in multiple repetition underwent a B-Z transition. In the case of plasmids containing AA/TT code words and substantial discontinuities in purine-pyrimidine alternation our analysis indicates that Z-Z junctions formed by A · T pairs contributed little to the overall energetic demands of the B-Z transition probably thanks to their high conformational flexibility.     

Altered replication in human cells promotes DMPK (CTG)(n) · (CAG)(n) repeat instability

Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n) · (CAG)(n) trinucleotide repeats (TNRs) in the 3' untranslated region (UTR) of the DMPK gene. Replication origins are cis-acting elements that potentiate TNR instability; therefore, we mapped replication initiation sites and prereplication complex protein binding within the ~10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, IS(DMPK) and IS(SIX5), flanked the (CTG)(n) · (CAG)(n) TNRs in control cells and in DM1 cells. Orc2 and Mcm4 bound near each of the replication initiation sites, but a dramatic change in (CTG)(n) · (CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n) · (CAG)(n) TNRs are cis-acting elements of instability in human cells, model cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)(n) · (CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n) · (CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n) · (CAG)(n) TNRs lead to replication stress. Consistent with this prediction, TNR instability increased in the HeLa model cells and DM1 cells upon small interfering RNA (siRNA) knockdown of the fork stabilization protein Claspin, Timeless, or Tipin. These results suggest that aberrant DNA replication and TNR instability are linked in DM1 cells.     

Structural studies on ligand–DNA systems: A robust approach in drug design

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.     

Bh-DNA: Variations of the Poly[d(A)] · Poly[d(T)] Structure within the Framework of the Fibre Diffraction Studies

Abstract

A refinement of the recent results for poly[d(A)] · poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4, 989 (1987)) involving additional parameters of the base-pair structure and of the sugar- phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)_n · d(T)_n stretch (Nelson et al., Nature 330, 221 (1987)).

Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)_n · d(T)_n sequence. The unique structural properties of poly [d(A)] · poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly [d(A)] · poly [d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11, 4141 (1983)) we suggest that this B-type structure be called B_h.     

Influence of Spermine on DNA Conformation in a Molecular Dynamics Trajectory of d(CGCGAATTCGCG)2: Major Groove Binding by One Spermine Molecule Delays the A→B Transition

Abstract

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)₂ has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A→B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A→B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like → B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B→A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.     

Nucleosides and Nucleotides. 105. DNA Bending in d(A)4 - d(T)4 TRACTS Containing 3-Deazaadenine or 7-Deazaadenine Substituted for Adenine§,1

Abstract

Self-complementary decadeoxyribonucleotides containing 3-deazaadenine or 7-deazaadenine in place of one of the adenine moieties in a parent sequence, d(GAAAATTTTC)_n, have been synthesized. After phosphorylation at their 5′-ends, the decamers were ligated to form multimers, which were analyzed by polyacrylamide gel electrophoresis. The multimers of the decamer containing 3-deazaadenine (3), d(GAA3ATTTTC)_n, showed decreased bending compared with the multimers of d(GAAAATTTTC)_n, while the decamer containing 3-deazaadenine at a different position, d(GAAA3TTTTC)_n, did not show any degree of bending. Also, the properties of migration of the multimers containing 7-deazaadenine in the gel will be discussed.