Evaluation of leaf water status by means of permittivity at terahertz frequencies

We present an electromagnetic model of plant leaves which describes their permittivity at terahertz frequencies. The complex permittivity is investigated as a function of the water content of the leaf. Our measurements on coffee leaves (Coffea arabica L.) demonstrate that the dielectric material parameters can be employed to determine the leaf water status and, therefore, to monitor drought stress in plant leaves. The electromagnetic model consists of an effective medium theory, which is implemented by a third order extension of the Landau, Lifshitz, Looyenga model. The influence of scattering becomes important at higher frequencies and is modeled by a Rayleigh roughness factor.     

Molecular crowding effects on structure and stability of DNA

Living cells contain a variety of biomolecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These biomolecules occupy a significant fraction (20-40%) of the cellular volume. The total concentration of biomolecules reaches 400gL(-1), leading to a crowded intracellular environment referred to as molecular crowding. Therefore, an understanding of the effects of molecular crowding conditions on biomolecules is important to broad research fields such as biochemical, medical, and pharmaceutical sciences. In this review, we describe molecular conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochemical and biophysical consequences of molecular crowding. Finally, we discuss the effect of molecular crowding on the structure, stability, and function of nucleic acids and the significance of molecular crowding in biotechnology and nanotechnology.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Changes of hydration during conformational transitions of DNA

Fiber X-ray diffraction and measurement of fiber dimensions yields information about the hydration of DNA in fibers. The results obtained give us the fraction of nucleotides in the B form for the A-B transition or the rate of progression for the B-C transition as functions of the number of water molecules per nucleotide. The present experimental results confirm the importance of cooperativity in the A-B transition and the progressive change of the DNA double helix conformation during the C-B transition. At least twenty additional water molecules per nucleotide are necessary to stabilize the B form for DNA molecules in fibers following the A to B transition whereas only ten are sufficient when the B conformation is obtained starting from the C form. Offprint requests to: S. Premilat     

Concentration-dependent effects of salicylaldoxime on chloroplast reactions

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (< 10 mM), salicylaldoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (< 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

The electrostatic field of the component units of DNA and its relationship to hydration

The electrostatic fields of the subunits of DNA are presented and compared with the corresponding electrostatic potentials. Differences are observed between these two properties, due to their different dependence on distance, which are of considerable interest since, whereas the potential may be used in studying the reactivity of molecules towards charged species the field can be a similar guide to attack by neutral dipolar molecules such as water. It is demonstrated, for the example of the purine and pyrimidine bases that the field may indeed be used to detect preferential hydration sites.     

Impact of the extrusion process on xanthan gum behaviour

Processing xanthan gum by extrusion and subsequent drying produces a biopolymer showing particulate, rather than molecular behaviour in aqueous solution. This form of xanthan disperses very readily to give a viscosity that is strongly dependent on salt concentration. On heating above the temperature of the order-disorder transition as determined by calorimetry, there is a viscosity transition that is indicative of the irreversible loss of the particulate structure. It is suggested that the extrusion process melts and aligns xanthan macromolecules. On cooling reordering will occur but in the highly concentrated environment in the extruder ( approximately 45% water w/w), inter-molecular association between neighbouring macromolecules cannot proceed to completion due to kinetic trapping. As a consequence a network structure is created maintained by associations involving ordered regions. A xanthan solution can be prepared from this particulate material by dispersing and subsequent heating far more readily than can be achieved with non-processed xanthan.     

Predicting allele frequencies in DNA pools using high density SNP genotyping data

To evaluate the ability to use DNA pools with the Illumina Infinium genotyping platform, two sets of gradient pools were created using two pairs of highly inbred chicken lines. Replicate pools containing 0%, 10%, 20%, 40%, 60%, 80%, 90% and 100% of DNA from line A vs. B or line C vs. D were created, for a total of 28 pools. All pools were genotyped for 12 046 SNPs. Three frequency estimation methods proposed in the literature (standard, heterozygote‐corrected and normalized) were compared with three alternate methods proposed herein based on mean square error (MSE), bias and variance of estimated vs. true allele frequencies and the fit of regression of estimated on true frequencies. The three new methods had average square root MSE of 4.6%, 4.6% and 4.7% compared to 5.2%, 5.5% and 11.2% for the three literature methods. Average absolute biases of the literature methods were 2.4%, 2.7% and 8.2% compared to 2.4% for all new methods. Standard deviations of estimates were also smaller for the new methods, at 3.1%, 3.2% and 3.2% compared to 3.5%, 4.0% and 5.0% for previously reported methods. In conclusion, intensity data from the Illumina Infinium Assay can be efficiently used to estimate allele frequencies in pools, in particular using any of the new methods proposed herein.     

Evaluating the effects of enhanced processivity and metal ions on translesion DNA replication catalyzed by the bacteriophage T4 DNA polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-1)). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn(2+) for Mg(2+) did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45.     

Sequence effects of single base loops in intramolecular quadruplex DNA

We have examined the properties of intramolecular G-quadruplexes in which the G3 tracts are separated by single base loops. The most stable complex contained 1',2'-dideoxyribose in all three loops, while loops containing T and C were slightly less stable (by about 2 degrees C). Quadruplexes containing loops with single A residues were less stable by 8 degrees C for each T to A substitution. These folded sequences display similar CD spectra, which are consistent with the formation of parallel stranded complexes with double-chain reversal loops. These results demonstrate that loop sequence, and not just length, affects quadruplex stability.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.     

Differential effects of cisplatin on mouse hepatic and renal mitochrondrial DNA

The objective of this study was to determine if the nephrotoxic effects induced by cisplatin were correlated to mitochondrial DNA damage. Comparisons were made with the liver since hepatotoxicity is rarely observed. Cisplatin doses of 10, 20 and 40 mg/kg were administered intraperitoneally to C57BL/6J mice. Mitochrondrial DNA was isolated from both the hepatic and renal tissues and quantitated by hybridization with a specific mitochondrial probe. Cisplatin caused differential effects on mouse hepatic and renal mitochondrial DNA. The 10 and 20 mg/kg dose caused an elevation in mitochondrial DNA levels in the hepatic, but no increase in the renal tissue was observed. This is the first study demonstrating an organ specific effect of cisplatin at the DNA level.     

DNA A-tracts bending: polarization effects on electrostatic interactions across their minor groove

Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.     

Global effects of DNA replication and DNA replication origin activity on eukaryotic gene expression

This report provides a global view of how gene expression is affected by DNA replication. We analyzed synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression. We use a higher‐order singular value decomposition to integrate the global mRNA expression measured in the multiple time courses, detect and remove experimental artifacts and identify significant combinations of patterns of expression variation across the genes, time points and conditions. We find that, first, ～88% of the global mRNA expression is independent of DNA replication. Second, the requirement of DNA replication for efficient histone gene expression is independent of conditions that elicit DNA damage checkpoint responses. Third, origin licensing decreases the expression of genes with origins near their 3′ ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.     

DNA Damage Tolerance and a Web of Connections with DNA Repair at Yale

This short article summarizes some of the research carried out recently by my laboratory colleagues on the function of DNA polymerase zeta (polζ) in mammalian cells. Some personal background is also described, relevant to research associations with Yale University and its continuing influence. Polζ is involved in the bypass of many DNA lesions by translesion DNA synthesis and is responsible for the majority of DNA damage-induced point mutagenesis in mammalian cells (including human cells), as well as in yeast. We also found that the absence of this enzyme leads to gross chromosomal instability in mammalian cells and increased spontaneous tumorigenesis in mice. Recently, we discovered a further unexpectedly critical role for polζ: it plays an essential role in allowing continued rapid proliferation of cells and tissues. These observations and others indicate that polζ engages frequently during DNA replication to bypass and tolerate DNA lesions or unusual DNA structures that are barriers for the normal DNA replication machinery.     

Checkpoint mechanisms at the intersection between DNA damage and repair

In response to genomic insults cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to help the cell to cope with the damage by coordinating cell cycle progression, DNA replication and DNA repair mechanisms. Accumulating evidence suggests that activation of the first checkpoint kinase in the cascade is not due to the lesion itself, but it requires recognition and initial processing of the lesion by a specific repair mechanism. Repair enzymes likely convert a variety of physically and chemically different lesions to a unique common structure, a ssDNA region, which is the checkpoint triggering signal. Checkpoint kinases can modify the activity of repair mechanisms, allowing for efficient repair, on one side, and modulating the generation of the ssDNA signal, on the other. This strategy may be important to allow the most effective repair and a prompt recovery from the damage condition. Interestingly, at least in some cases, if the damage level is low enough the cell can deal with the lesions and it does not need to activate the checkpoint response. On the other hand if damage level is high or if the lesions are not rapidly repairable, checkpoint mechanisms become important for cell survival and preservation of genome integrity.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

Local deformations revealed by dynamics simulations of DNA polymerase Beta with DNA mismatches at the primer terminus

Nanosecond dynamics simulations for DNA polymerase beta (pol beta)/DNA complexes with three mismatched base-pairs, namely GG, CA, or CC (primer/template) at the DNA polymerase active site, are performed to investigate the mechanism of polymerase opening and how the mispairs may affect the DNA extension step; these trajectories are compared to the behavior of a pol beta/DNA complex with the correct GC base-pair, and assessed with the aid of targeted molecular dynamics (TMD) simulations of all systems from the closed to the open enzyme state. DNA polymerase conformational changes (subdomain closing and opening) have been suggested to play a critical role in DNA synthesis fidelity, since these changes are associated with the formation of the substrate-binding pocket for the nascent base-pair. Here we observe different large C-terminal subdomain (thumb) opening motions in the simulations of pol beta with GC versus GG base-pairs. Whereas the conformation of pol beta in the former approaches the observed open state in the crystal structures, the enzyme in the latter does not. Analyses of the motions of active-site protein/DNA residues help explain these differences. Interestingly, rotation of Arg258 toward Asp192, which coordinates both active-site metal ions in the closed "active" complex, occurs rapidly in the GG simulation. We have previously suggested that this rotation is a key slow step in the closed to open transition. TMD simulations also point to a unique pathway for Arg258 rotation in the GG mispair complex. Simulations of the mismatched systems also reveal distorted geometries in the active site of all the mispair complexes examined. The hierarchy of the distortions (GG>CC>CA) parallels the experimentally deduced inability of pol beta to extend these mispairs. Such local distortions would be expected to cause inefficient DNA extension and polymerase dissociation and thereby might lead to proofreading by an extrinsic exonuclease. Thus, our studies on the dynamics of pol beta opening in mismatch systems provide structural and dynamic insights to explain experimental results regarding inefficient DNA extension following misincorporation; these details shed light on how proofreading may be invoked by the abnormal active-site geometry.