Charge Transport in Poly(dG)–Poly(dC) and Poly(dA)–Poly(dT) DNA Polymers

We investigate the charge transport in synthetic DNA polymers built up from single type of base pairs. In the context of a polaronlike model, for which an electronic tight-binding system and bond vibrations of the double helix are coupled, we present estimates for the electron-vibration coupling strengths utilizing a quantum-chemical procedure. Subsequent studies concerning the mobility of polaron solutions, representing the state of a localized charge in unison with its associated helix deformation, show that the system for poly(dG)–poly(dC) and poly(dA)–poly(dT) DNA polymers, respectively possess quantitatively distinct transport properties. While the former supports unidirectionally moving electron breathers attributed to highly efficient long-range conductivity, the breather mobility in the latter case is comparatively restrained, inhibiting charge transport. Our results are in agreement with recent experimental results demonstrating that poly(dG)–poly(dC) DNA molecules acts as a semiconducting nanowire and exhibit better conductance than poly(dA)–poly(dT) ones.     

Two Binding Modes of Netropsin are Involved in the Complex Formation with Poly(dA-dT)·Poly(dA-dT) and other Alternating DNA Duplex Polymers

Abstract

Using CD measurements we show that the interaction of netropsin to poly(dA-dT)·poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA·dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA)·poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT)·poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

Structure of Poly (dT)·Poly (dA)·Poly (dT)

Abstract

The molecular structure of poly (dT)·poly (dA)·poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right- handed triple-helix of pitch 38.4 Å and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2′-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

Structure of Poly(dA)·Poly(dT) is not Identical to the AT Rich Regions of the Single Crystal Structure of CGCGAATTBrCGCG. The Consequence of this to Netropsin Binding to Poly(dA)·Poly(dT)

Abstract

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA)·poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA)·poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA)·poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations—dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA)·poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Monte-Carlo Simulation of DNA Duplex Hydration. B and B′ Conformations of Poly(dA) · Poly(dT) Have Different Hydration Shells

Abstract

Monte-Carlo simulation of poly(dA) · poly(dT) hydration by 30 water molecules per nucleotide pair has been performed. Two B-family conformations, both with a 36° helical twist but with different minor groove widths, were considered. One conformation is Arnott's standard B form, the other one is specific for poly(dA) · poly(dT) B′ form with a narrowed minor groove. The mean energies and the mean numbers of water-water and water-DNA hydrogen bonds are close for the two conformations. Nevertheless, the hydration shell of the B' form differs drastically from that of the standard B form. The water arrangement in the minor groove of the B′ form resembles the spine of hydration in the central part of Dickerson's dodecamer d(CGCGAATTCGCG). No such spine is formed in the hydration shell of the usual B form with a wider minor groove. In this conformation water bridges between adenine N3 or thymine O2 and oxygen of the sugar ring of the neighbouring nucleotide along the chain can be formed (“strings” in Dickerson's decamer d(CCAAGATTGG)).     

Poly(ADP-ribose) Polymerase-1 Inhibits Strand-Displacement Synthesis of DNA Catalyzed by DNA Polymerase β

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dA-dT) poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni²⁺ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl₂ in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm″^?1coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm^?1 region characterizing local chemical interactions of the Ni²⁺ ions with the adenien N7 position, iii) lines at about 1483-and 1582 cm^?1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680-and 1733 cm^?1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT) poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Chromosome numbers in selectedHieracium species in the Krkonoše Mts. (the West Sudeten)

Chromosome numbers are given of 15 species of the genusHieracium L. s. str., representing seven species groups (in the sense of Flora Europaea, roughly corresponding to Zahn's “species principales”) from the Krkono?e Mts., N. Bohemia and SW Poland. For the first time chromosome numbers are reported forH. melanocephalum Tausch (2n=27),H. tubulosum Tausch (2n=36),H. schustleri Zlatník (2n=36),H. fritzei F. Schultz (2n=27),H. rohlenae Zlatník (2n=27),H. nigrescens Willd. (2n=36),H. decipiens Tausch (2n=36),H. atrellum Juxip inSchischkin etBobrov (2n=27),H. subnigrescens (Fries exNorrlin)Dahlst. (2n=36),H. sudeticum Sternberg (2n=36),H. pedunculare Tausch (2n=36),H. glandulosodentatum Uechtr. (2n=36),H. wimmeri Uechtr. (2n=27). InHieracium alpinum L. s. str. the number 2n=27 has been confirmed. The results show a high proportion of tetraploid taxa; no diploids have been found.     

The 3′–5′ DNA Exonuclease TREX1 Directly Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) during the DNA Damage Response

The main function of the 3′–5′ DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations.     

DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.     

Parastenocaris andreji n. sp. (Crustacea; Copepoda) – the first record of the genus in Slovenia (SE Europe)

Parastenocaris andreji n. sp. was found in ground water in a northwestern part of Slovenia (southern part of Central Europe). Two adult females of the new species were filtered out of 20 000 l of water during examination of an aquifer for water supply. The bore well was 10 m deep. The aquifer is fed by rainwater and probably only occasionally from a nearby alpine river Sava. The new species differs markedly from other known species of the genus by shape and armature of furcal rami, very long and slim maxillae and some characters on endopodites P2–P5. The closest related species are probably Parastenocaris nolliKiefer, 1938 with subspecies P. nolli alpinaKiefer, 1960, P. gertrudae Kiefer, 1968 and P. austriacaKiefer, 1976.     

Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.     

Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

Absorption of DNA in the region of vacuum-uv (3–25 eV)

By means of a device that might be considered a modern version of "Ulbricht's sphere" the absorption spectrum and the photoelectric emission of calf thymus DNA was measured in the region of 3 to 25 eV (400 to 50 nm). A tentative explanation of the general shape of the absorption spectrum and of its 6 maxima is given. The results permit a much better insight into some biologic effects of vacuum-uv to be gained than hitherto possible.     

Chromosome numbers and DNA?C values in the genus Lippia (Verbenaceae)

The genus Lippia comprises herbs, shrubs, and small trees, including many species with medicinal properties. The species are distributed throughout South and Central America and Tropical Africa, but the majority of them occur in Brazil, Paraguay, and Argentina. The DNA?C value of 28 Brazilian species has been estimated by flow cytometry. Estimated DNA?C values ranged from 0.825?pg (L. corymbosa) to 2.150?pg (L.?brasiliensis). In addition, new chromosome numbers of 12 species have also been described, and meiotic cells with 12, 13, and 14 chromosome pairs were observed. A straightforward correlation between chromosome number and DNA?C value was not observed, probably due to two outlier species of Lippia that have been transferred from the genus Lantana. In general, the data confirm previous reports regarding the variation within the taxonomic sections and also suggest a new revision in section Zapania. Aspects of karyotypic evolution of the genus are also discussed.