Antiparallel DNA duplex formation between alternating alpha d(GA)n and beta d(GA)n sequences

In the Adult Intensive Care Unit of The Prince of Wales Hospital, Sydney, Australia, inspiratory gas is humidified to saturation at 37 degrees C. This stops the buildup of dried sputum within the endotracheal tubes and thus prevents blocked tubes and the increased resistance caused by partial obstruction. Inspiratory and expiratory hose heater wires are used to produce a completely dry circuit, allowing the elimination of water traps and circuit support arms without the resistance of a heat and moisture exchanger.     

Small ligands that neither bind to nor alter the structure of d(GA x TC)n sequences in DNA

Three minor-groove binding ligands have been used to study the characteristics of two d(GA x CT)n DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia-Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA x CT)5 nor to d(GA x CT)22 extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA x CT)n flanking sequences located near DNA regions where the drug was bound. The d(GA x CT)n sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.     

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.     

Molecular dynamics studies of axis bending in d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5): effects of sequence polarity on DNA curvature

Gel retardation studies and other experiments indicate that DNA sequences containing the d(GA4T4C)n motif are curved, whereas those of identical composition but with a reverse sequence polarity, the d(GT4A4C)n motif, are straight. Hydroxyl radical cleavage experiments show that d(GA4T4C)n shows a unique signature, whereas d(GT4A4C)n behaves normally. To explain these results at a molecular level, molecular dynamics (MD) simulations were performed on the DNA duplexes d(G5-(GA4T4C)2-C5) and d(G5-(GT4A4C)2-C5) to 3.0 and 2.5 ns, respectively. The MD simulations are based on the Cornell force field implemented in the AMBER 4.1 modeling package and performed in a neutral solution of anionic DNA with K+, Cl- and Mg2+ at concentrations roughly comparable to a ligase buffer. Long range interactions were treated by the particle mesh Ewald method. Analysis of the results shows that the calculated dynamical structure of d(G5-(GA4T4C)2-C5) exhibits strong gross curvature, consistent with the observed behavior. The most significant locus of curvature in the MD structure is found at the central C15-G16 step, with an average roll angle of 12.8(+/-6.40)deg. The d(G5-(GT4A4C)2-C5) MD structure exhibited significantly less gross curvature. Analysis of results indicates that the reduction in gross curvature in the d(G5-(GT4A4C)2-C5) trajectory originates from the effect of the T10-A11 and T20-A21 steps, which showed average roll angles of 12.5(+/-5)deg. These three steps, T10-A11, C15-G16 and T20-A21, are half-helix turns away from one another, and their contributions to concerted bending cancel out. The A-tracts in the MD structure are essentially straight. The dynamical structure of d(G5-(GA4T4C)2-C5) exhibited minor groove deformation comprised of expansion at the 5' end of A-tracts and progressive narrowing towards the 3' end, consistent with and elaborating the interpretation of hydroxyl radical chemical probing results.     

Effects of the introduction of L-nucleotides into DNA. Solution structure of the heterochiral duplex d(G-C-G-(L)T-G-C-G).d(C-G-C-A-C-G-C) studied by NMR spectroscopy

The effect of the substitution of a L-nucleoside for a D-nucleoside in the duplex d(G-C-G-T-G-C-G).d(C-G-C-A-C-G-C) was studied by UV and NMR spectroscopy. These unnatural oligonucleotides have potential for antisense DNA technology [Damha, M. J., Giannaris, P. A., & Marfey, P. (1994) Biochemistry (preceding paper in this issue)]. The thermal stability of such duplexes is lower than that of the natural one and is dependent on the nucleotide type and/or sequence. Interestingly, inversion of the chirality of thymidine but not adenosine coincides with a large stabilizing enthalpy change. The structure of the heterochiral duplex d(G1-C2-G3-(L)T4-G5-C6-G7).d(C8-G9-C10-A11-C12-G13- C14), where (L)T denotes the mirror image of the natural thymidine, has been determined by NMR spectroscopy. The sugar conformation was determined using the sum of coupling constants and the distances using a model free relaxation matrix approach. The torsion angles of the backbone follow from 3JHH, 3JHP, and 4JHP coupling constants. The structure of the duplex was calculated by metric matrix distance geometry followed by simulated annealing. The structure is close to that of B-DNA. The base pair formed by (L)T and A is of the Watson-Crick type. All sugars adopt an S-type pucker. The incorporation of the L-sugar in the duplex is accomplished by changes in the backbone torsion angles around the phosphates and the glycosidic torsion angle of (L)T. The modification induces changes in the natural strand as well. The structure exhibits an unusual interaction between the aromatic rings of the (L)T4.A11 and G3.C12 base pairs, which provides a plausible explanation of the unusual thermodynamic properties of the duplex.     

Binding of anti-double stranded (ds) DNA-positive sera to denatured (d) DNA and synthetic poly[dA-dT] x poly[dA-dT] double stranded copolymer in an ELISA format

Full dose heparin therapy is monitored by a variety of laboratory methods, of which the activated partial thromboplastin time (APTT) is the most popular. A large number of APTT reagents are currently available, with different sensitivities to heparin evident in many. Within the literature it is apparent that there is a lack of consensus, and indeed some confusion, regarding the therapeutic ranges for the APTT for standard heparin therapy in the treatment of venous thromboembolic disease. Accordingly we conducted an Australasian survey to evaluate current laboratory and clinical practices in monitoring heparin therapy, to determine the extent of variation in the approach and to stimulate the process of standardisation of acceptable procedures and methodology. Results of the survey demonstrate that currently there is no uniform practice used to establish therapeutic ranges for monitoring standard heparin therapy. Furthermore, results suggest that current practice may lead to subtherapeutic anticoagulation in many laboratories.     

PARODS--a study of parallel algorithms for ordering DNA sequences

Based on assumptions about the pathophysiology of egg-related lesions in the lower reproductive tract, putative indirect disease markers were investigated in vaginal fluids from 54 Malawi adolescent girls and women infected with S. haematobium. These women received a careful gynecological examination during which biopsies were taken from the cervix, and, if present, also from suspicious lesions in the vagina and the vulva. If the biopsies, either in wet crushed preparations or in histological sections, contained eggs the patients were considered to have female genital schistosomiasis (FGS; n = 33). The remainder (n = 21) were classified as having urinary schistosomiasis only. Eosinophil cationic protein (ECP), a cytotoxic granule protein of eosinophils, neopterin, a second messenger molecule generated during the activation of macrophages, and IgA as an indicator of local B-cell activation were quantitatively determined in vaginal fluid. To clarify the origin of ECP, this protein was also looked for in histological sections by an immunohistochemical method. In order to explore whether such disease markers can be detected after absorption to a tampon-like material, ECP and IgA were also assessed after elution from a non-porous, polypropylene fibre web impregnated with vaginal fluid. The concentration of ECP in vaginal fluid and the degree of immunohistochemical staining in histological sections were significantly higher in patients with FGS than in women with urinary schistosomiasis only. The amount of ECP detected in histological sections correlated to the number of eggs/mm2 of compressed genital tissue (rho = 0.36, P = 0.02), and the concentration of ECP in vaginal fluid correlated to the concentration of neopterin as well as to that of IgA (rho = 0.52, P = 0.004 and rho = 0.37, P = 0.02, respectively). Median neopterin concentration in vaginal fluid was also higher in the FGS group, but the difference was not statistically significant. ECP could also be detected in eluates from impregnated fibre webs, but the concentration was approximately one power of 10 less than in the original vaginal fluid. These results demonstrate that indicators of immunological mechanisms related to the egg-granuloma might be useful as indirect disease markers for women with FGS if assessed in vaginal washings or swab eluates.     

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.     

Effect of flanking sequences on the right- to left-handed transition of a (dA-dT)n tract in supercoiled DNA

Alternating (dA-dT)n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni2+ ions and high NaCl concentration (Nejedly, K., Klysik, J. and Palecek, E., FEBS Lett. 243, 313-317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)n tract. In particular, the presence of 5'-homopyrimidine (C3) and 3'-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left-handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT)n sequence.     

Structure of poly d(AI).poly d(CT) in two different packing arrangements

X-ray diffraction analysis of poly d(AI).poly d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 A and 32.4 A in the two cases, almost 1.5 A shorter than in classical B-DNA. The resulting B'-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3.0 A resolution.     

Crystal structures of the side-by-side binding of distamycin to AT-containing DNA octamers d(ICITACIC) and d(ICATATIC)

To understand the recognition interactions between AT-containing alternating DNA and minor groove binding drugs, the crystal structures of the side-by-side binding of two distamycin molecules to the DNA octamers d(ICITACIC)2 and d(ICATATIC)2, referred to here as TA and ATAT, respectively, have been determined at 1.6 A and 2.2 A, respectively. Compared to the previous 2:1 all-IC d(ICICICIC)2-distamycin complex, the substitutions of the I x C base-pairs by the A x T base-pairs enable the interactions of the drug with its natural target to be studied. Both complexes assume side-by-side drug binding, isomorphous to the all IC counterpart in the tetragonal space group P4(1)22 (a = b = 28.03 A, c = 58.04 A and a = b = 27.86 A, c = 58.62 A, respectively). The ATAT complex also crystallized in a new polymorphic monoclinic space group C2 (a = 33.38 A, b = 25.33 A, c = 28.11 A and beta = 120.45 degrees) and was solved at 1.9 A resolution. The structures of the three double drug x DNA complexes are very similar, characterized by systematic hydrogen bonding and van der Waals interactions. Each drug hydrogen bonds with the bases of the proximal DNA strand only and stacks with the sugar moiety, while the side-by-side drugs themselves exhibit pyrrole ring-peptide stacking. The pyrrole-peptide interaction is crucial for the side-by-side binding mode of the distamycin/netropsin family of drugs. The purine-pyrimidine alternation is probably responsible for the striking alternation in the helical and backbone conformations. The structures are conserved between the pure IC complex and the AT substituted complexes but further details of the side-by-side binding to DNA are provided by the 1.6 A resolution structure of TA.     

C4d DNA sequences of two infrequent human allotypes (C4A13 and C4B12) and the presence of signal sequences enhancing recombination

The DNA sequences of the polymorphic region (C4d) that belong to the infrequent complement C4 allotypes C4A13 and C4B12 have been obtained. In addition, C4A4 and C4B2 C4d sequences have been completed. C4A13 shows a new combination of amino acids at the following polymorphic positions: Asp1054, Pro1101 Cys1102, Leu1105, Asp1106, Asn1157, Ala1188, and Arg1191. These amino acids conform to the antigenic determinants Chido 1 and Rodgers 3; thus C4A13 is the only allele described thus far that carries both Ags. C4A13 and C4A4 carry the motif "ggctc*" (* means "deletion") at positions 14 to 19 in their intron 28; this motif had previously been reported only in C4B alleles. The C4B12 nucleotide sequence is analogous to C4B1b and C4B3 sequences, except for codon 1076, which is GCC in C4B1b and C4B3 and GGA in C4B12, which is coding for glycine in both cases. A recombination model for the generation of C4 alleles is formulated based on the analysis of these new sequences. One recombination would take place between positions 1157 and 1186 and would give rise to C4A13 and C4B5 or C4A3 (or C4A6) and C4B2; another one would occur between positions 1054 and 1076 and would generate C4A3 (or C4A6) and C4B12 or C4A2 and C4Bnew. Analysis of 1157 to 1186 and 1054 to 1076 fragments reveals the presence of putative sequence signals for recombination (similar to Escherichia coli chi recombination signal); the accumulation of such signals in fragments 1054 to 1076 supports the notion that a recombination hot spot for the C4 gene may exist and it also enhances new allele generation and intraspecies C4 gene homogenization.     

Structure of a 16-mer RNA duplex r(GCAGACUUAAAUCUGC)2 with wobble C.A+ mismatches

The crystal structure of a 16-mer, the longest known RNA duplex, has been determined at 2.5 A resolution. The hexadecamer r(GCAGACUUAAAUCUGC) contains isolated C.A/A.C mismatches with two hydrogen bonds. The two hydrogen bonds in the mismatches suggests that N1 of A is protonated even though the crystallization was done at neutral pH. Therefore, the C.A mismatch is a C.A+ wobble similar to the G.U wobble. The two C.A+ pairs are isolated by four Watson-Crick pairs and flanked by five Watson-Crick base-pairs on either sides. Kinks/bends of 20 degrees are observed at the wobble sites. The Watson-Crick base-pair A5.U26 on the 5'-side of the first C6.A27(+) wobble has a twist angle of 27 degrees compared to the 3'-side U7.A28 pair of 36 degrees. The twist angles are reversed (37 degrees and 26 degrees) in the second A11(+).C22 wobble because of the approximate dyad in the molecule, the flanking base-pair sequences are A.U pairs. The wobbles expand the major groove to 7.1 A/7.3 A. The duplexes form helical columns and are tightly packed around the 31-screw axis. The minor grooves of adjacent columns in juxtaposition interact through the O2' atoms and the anionic phosphate oxygen atoms.