Molecular and Crystal Structure of d(CGCGmo4CG): N4−Methoxy-cytosineguanine Base Pairs in Z-DNA

Abstract

The base analogue N⁴?methoxycytosine (mo⁴C) is ambivalent in its hydrogen-bonding potential. In d(CGCGmo⁴CG) it is in the imino form and so mimics thymine when wobble base pairing with guanine.     

Ab Initio Quantum Mechanics Analysis of Imidazole C-H…O Water Hydrogen Bonding and a Molecular Mechanics Forcefield Correction

Abstract

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but ‘activated’ C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H…O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H…0 and C—O. In general, these calculations indicate that C-H…0 interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) level of theory of a water C-H…O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H…O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole C_e?H…O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole C_e?H…O water and imidazole N_d?H…O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H…O water interaction, these forcefields do not adequately account for the imidazole C_e?H…O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H…O hydrogen bonded complex.     

A Systematic Analysis of Buried Polar Side Chains and Their Interactions in α-Helical Proteins

Examination of 80 -helical proteins and domains demonstrates that they contain from 1 to more than 20 completely buried (water-inaccessible) polar side chains. As a rule the latter have partners for H-bonding but the resulting H-bond system is often not saturating. Basing on statistical analysis, we determined the optimal number of H-bonds for every type of polar side chain, and discuss the structural role of vacant donors and acceptors. About half of the H-bonds formed by buried side chains pertain to interhelix contacts of the (side chain)–(side chain) and (side chain)–(main chain) types. Such interactions appear to be a most important factor determining the mutual arrangement of -helices in proteins. Analysis of the frequency of occurrence of various interacting pairs reveals that these interactions are selective.     

Pattern of glacial—interglacial vegetation in subtropical Chile

Pleistocene vegetation in the Mediterranean climate region of subtropical Chile during >40,000 yr of the last glaciation stands in contrast with vegetation of the past 10,000 yr of the present interglaciation. During the last ice age, open woodland of southern beech (Nothofagus dombeyi and N. obliqua types) and Andean podocarp (Prumnopitys andina) mixed with grasses and composites, was established on unglaciated, low‐lying terrain now occupied by broad sclerophyllous plant communities. This marked vegetation change during a glacial‐interglacial cycle, inferred from pollen contained in a core from Laguna de Tagua Tagua (34°30'S), suggests a pattern applicable to the vegetational setting during repeated cycles throughout the Quaternary.

Ice‐age vegetation can be accounted for by year‐long domination of the polar maritime air mass at lower, middle latitudes, unlike modern interglacial type plant formations, which are regulated by seasonal interaction between subtropical and polar maritime air. Glacial‐interglacial shifting of air mass centers, as a cause for the changing distribution of species and plant communities, appears geared to events in the atmosphere/ocean system.     

Analysis of plastid and nuclear DNA data in plant phylogenetics——evaluation and improvement

Correct combination of plastid(cp)and nuclear(nr)DNA data for plant phylogenetic reconstructions is not a new issue,but with an increasing number of nrDNA loci being used,it is of ever greater practical concern.For accurately reconstructing the phylogeny and evolutionary history of plant groups,correct treatment of phylogenetic incongruence is a vital step in the proper analysis of cpDNA and nrDNA data.We first evaluated the current status of analyzing cpDNA and nrDNA data by searching all articles published in the journal Systematic Botany between 2005 and 2011.Many studies combining cpDNA and nrDNA data did not rigorously assess the combinability of the data sets,or did not address in detail possible reasons for incongruence between the two data sets.By reviewing various methods,we outline a procedure to more accurately analyze and/or combine cpDNA and nrDNA data,which includes four steps:identifying significant incongruence,determining conflicting taxa,providing possible interpretations for incongruence,and reconstructing the phylogeny after treating incongruence.Particular attention is given to explanation of the cause of incongruence.We hope that our procedure will help raise awareness of the importance of rigorous analysis and help identify the cause of incongruence before combining cpDNA and nrDNA data.     

The role of OH…O and CH…O hydrogen bonds and H…H interactions in ethanol/methanol–water heterohexamers

Bioethanol is one of the world’s most extensively produced biofuels. However, it is difficult to purify due to the formation of the ethanol–water azeotrope. Knowledge of the azeotrope structure at the molecular level can help to improve existing purification methods. In order to achieve a better understanding of this azeotrope structure, the characterization of (ethanol)₅–water heterohexamers was carried out by analyzing the results of electronic structure calculations performed at the B3LYP/6-31+G(d) level. Hexamerization energies were found to range between ?36.8 and ?25.8 kcal/mol. Topological analysis of the electron density confirmed the existence of primary (OH…O) hydrogen bonds (HBs), secondary (CH…O) HBs, and H…H interactions in these clusters. Comparison with three different solvated alcohol systems featuring the same types of atom–atom interactions permitted the following order of stability to be determined: (methanol)₅–water > (methanol)₆ > (ethanol)₅–water > (ethanol)₆. These findings, together with accompanying geometric and spectroscopic analyses, show that similar cooperative effects exist among the primary HBs for structures with the same arrangement of primary HBs, regardless of the nature of the molecules involved. This result provides an indication that the molecular ratio can be considered to determine the unusual behavior of the ethanol–water system. The investigation also highlights the presence of several types of weak interaction in addition to primary HBs.

Open image in new window

Graphical Abstract Water-ethanol clusters exhibit a variety of interaction types between their atoms, such as primary OH...O (blue), secondary CH...O (green) and H...H (yellow) interactions as revealed by Quantum Chemical Topology

     

The Concerted Action of Msh2 and UNG Stimulates Somatic Hypermutation at A · T Base Pairs

Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A·T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase η in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A·T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A·T mutations. Our analysis further indicates that repair tracts typically are ∼200 nucleotides long and that polymerase η makes ∼1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A·T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.The affinity maturation of the antibody response depends on the somatic hypermutation (SHM) process. The enzyme activation-induced cytidine deaminase (AID) initiates SHM in germinal center B cells by deaminating C within immunoglobulin (Ig) genes, yielding a G·U lesion that is resolved by several mechanisms (29). Replication across the U generates G·C to A·T transition mutations, while the removal of the U by uracil-N-glycolase (UNG) leads to transversion and transition mutations at the original G·C base pair (33). The AID-generated G·U lesion is also a substrate for the mismatch repair (MMR) proteins Msh2, Msh6, and Exo1. Unlike their normal role in DNA repair, the processing of this lesion by these MMR proteins during SHM paradoxically leads to the production of mutations at A·T base pairs (see below).MMR is a DNA repair process utilized by prokaryotes and eukaryotes (25). This pathway repairs DNA errors caused by the misincorporation of nucleotides during DNA synthesis. The initial mismatch is detected by MutSα, which consists of Msh2 and Msh6 in mammalian cells. The ability of MMR to discriminate between the mutated and unmutated strands of DNA is thought to be dictated by nicks or gaps on the newly synthesized lagging strand between Okazaki fragments or by strand ends on the leading strand at the replication fork (18). The MutLα endonuclease (Mlh1/Pms2) uses the DNA nick or end as a marker of the newly synthesized, and therefore mutated, strand to introduce a new nick on either side of the mismatch (15). This nicked strand is then excised by the 5′-to-3′ exonuclease Exo1, and the ensuing gap is repaired by the replicative polymerase δ. However, since AID acts primarily during G₁ of the cell cycle (11, 36), it is unclear whether Msh2/6 is capable of distinguishing between the AID-mutated and unmutated strands prior to strand excision.Consistently with their role in DNA repair, deficiency in Msh2, Msh6, or Exo1 generally leads to an increase in mutation frequencies in different tissues (40). However, in the case of SHM of Ig genes, the loss of these MMR proteins reduces the frequency of mutations at A·T base pairs (4, 5, 10, 16, 22, 30, 32, 37, 41, 42). One possible difference between conventional and mutagenic MMR is the involvement of the error-prone DNA polymerase η in the latter process. Indeed, both mice and humans lacking polymerase η resemble Msh2-deficient mice, in that mutations at A·T base pairs in the V region are less frequent (6, 7, 47). Moreover, the error spectrum of polymerase η on undamaged DNA matches the mutation spectrum of A·T mutations in the V region (35). While it is now well established that mutations at A·T base pairs are produced largely by proteins involved in the MMR pathway, it is not known how DNA polymerase η is recruited during SHM.One possible explanation for the use of error-prone polymerases is the occurrence of replication-blocking lesions, such as an abasic site or a modified nucleotide, in the V region of Ig genes. Evidence that a replication block leads to mutagenic MMR comes from recent studies showing the requirement of ubiquitinated PCNA for mutagenic MMR (1, 19, 34). Monoubiquitination at the K164 residue of PCNA in response to DNA damage leads to translesional synthesis (1), and SHM at A·T base pairs is reduced in PCNA^K164R/K164R mice to levels observed in MMR-deficient mice (19, 34). In addition, the finding that translesional DNA polymerases are involved in SHM (9, 31, 45-47) suggests that replication-blocking lesions are common at the Ig locus during SHM. Taken together, these observations suggest a model in which replication-blocking lesions recruit error-prone polymerases, which then generate mutations at nearby A·T base pairs. As reported here, we have tested this model by examining the correlated mutations in V region sequences from hypermutating Ramos cells and in murine centroblasts.     

Base recognition of gap sites in DNA–DNA and DNA–RNA duplexes by short oligonucleotides

To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4 nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites.     

Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.     

Base Composition and Hybridization Studies of the Three Double-Stranded RNA Segments of Bacteriophage φ6

The three double-stranded ribonucleic acid (dsRNA) segments of the bacteriophage phi6 were isolated and shown to have similar melting temperatures and base compositions. RNA:RNA hybridization experiments with the isolated segments eliminate the possibility that the two smaller dsRNA segments arise from a cleavage of the large dsRNA segment. The two smaller RNA segments reanneal rapidly even at low temperatures; in contrast, the large dsRNA reannealed only at higher temperatures. Evidence is also presented which suggests that the dsRNAs may contain a short single-stranded RNA tail.     

Analysis of protein carbonylation — pitfalls and promise in commonly used methods

Abstract

Oxidation of proteins has received a lot of attention in the last decades due to the fact that they have been shown to accumulate and to be implicated in the progression and the pathophysiology of several diseases such as Alzheimer, coronary heart diseases, etc. This has also resulted in the fact that research scientists are becoming more eager to be able to measure accurately the level of oxidized protein in biological materials, and to determine the precise site of the oxidative attack on the protein, in order to get insights into the molecular mechanisms involved in the progression of diseases. Several methods for measuring protein carbonylation have been implemented in different laboratories around the world. However, to date no methods prevail as the most accurate, reliable, and robust. The present paper aims at giving an overview of the common methods used to determine protein carbonylation in biological material as well as to highlight the limitations and the potential. The ultimate goal is to give quick tips for a rapid decision making when a method has to be selected and taking into consideration the advantage and drawback of the methods.     

Structure Versus Stochasticity—The Role of Molecular Crowding and Intrinsic Disorder in Membrane Fission

Cellular membranes must undergo remodeling to facilitate critical functions including membrane trafficking, organelle biogenesis, and cell division. An essential step in membrane remodeling is membrane fission, in which an initially continuous membrane surface is divided into multiple, separate compartments. The established view has been that membrane fission requires proteins with conserved structural features such as helical scaffolds, hydrophobic insertions, and polymerized assemblies. In this review, we discuss these structure-based fission mechanisms and highlight recent findings from several groups that support an alternative, structure-independent mechanism of membrane fission. This mechanism relies on lateral collisions among crowded, membrane-bound proteins to generate sufficient steric pressure to drive membrane vesiculation. As a stochastic process, this mechanism contrasts with the paradigm that deterministic protein structures are required to drive fission, raising the prospect that many more proteins may participate in fission than previously thought. Paradoxically, our recent work suggests that intrinsically disordered domains may be among the most potent drivers of membrane fission, owing to their large hydrodynamic radii and substantial chain entropy. This stochastic view of fission also suggests new roles for the structure-based fission proteins. Specifically, we hypothesize that in addition to driving fission directly, the canonical fission machines may facilitate the enrichment and organization of bulky disordered protein domains in order to promote membrane fission by locally amplifying protein crowding.     

Mode of action of N,N′-diphenylurea: The isolation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N,N′-diphenylurea

Summary The tRNA from cytokinin-dependent tobacco callus (Nicotiana tabacum) grown on mineral medium containing N,N-diphenylurea as the source of cytokinin was found to contain 3 cytokinin-active ribonucleosides. The 2 ribonucleosides present in the largest amounts were identified conclusively by their chromatographic properties, ultra-violet and low-resolution mass spectra as the naturally-occurring cytokinins 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine and 6-(3-methyl-2-butenylamino)-9--ribofuranosylpurine. A third ribonucleoside, present in smaller amounts, was identified as another naturally-occurring cytokinin 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9--D-ribofuranosylpurine on the basis of its chromatographic behaviour. No evidence was found to associate the mode of action of the non-purine cytokinin, N,N-diphenylurea, with tRNA.Abbreviation DPU N,N-diphenylurea     

N2O emissions from low and moderately disturbed pasture soils—field tests of minimal and maximal N supply

Rates and patterns of nitrogen transformation differ in divergently managed pasture soils. In pastures with low nutrient inputs, N is utilized efficiently and it is assimilated by plants and soil microorganisms for synthesis of biomass. In more intensive pastures, characterized with higher N inputs, significant amounts of N can be lost from the ecosystem in various forms. Two soils of a cattle overwintering area with different levels of cattle disturbance were supplied with a solution of KNO₃ in various levels corresponding in range to 0–500 kg N ha^?1. Emissions of N₂O were measured during 24 h after a NO₃ ^?-N application. We hypothesized that under a low disturbance small additions of up to 5 kg NO₃ ^?-N are used by plants and soil microbes without an increase in N₂O emissions, while a pasture adapted to a moderate disturbance will increase N₂O emissions. Results showed that in both soils, the addition of N always increased N₂O emissions, while emissions were more pronounced in soil at the location with a higher intensity of cattle traffic. Contrary to our hypothesis, however, NO₃ ^––N was not fully metabolized in the soil with low disturbance by the cattle. Probable explanations of such a result were lower intensity of N transformations in this soil and low utilisation of N by grass. Our results suggest that under certain conditions relatively low nitrate-N inputs can also stimulate N₂O fluxes from soils.     

Pattern and variation of C:N:P ratios in China’s soils: a synthesis of observational data

Inspired by previous studies that have indicated consistent or even well-constrained (relatively low variability) relations among carbon (C), nitrogen (N) and phosphorus (P) in soils, we have endeavored to explore general soil C:N:P ratios in China on a national scale, as well as the changing patterns of these ratios with soil depth, developmental stages and climate; we also attempted to determine if well-constrained C:N:P stoichiometrical ratios exist in China’s soil. Based on an inventory data set of 2,384 soil profiles, our analysis indicated that the mean C:N, C:P and N:P ratios for the entire soil depth (as deep as 250 cm for some soil profiles) in China were 11.9, 61 and 5.2, respectively, showing a C:N:P ratio of ~60:5:1. C:N ratios showed relatively small variation among different climatic zones, soil orders, soil depth and weathering stages, while C:P and N:P ratios showed a high spatial heterogeneity and large variations in different climatic zones, soil orders, soil depth and weathering stages. No well-constrained C:N:P ratios were found for the entire soil depth in China. However, for the 0–10 cm organic-rich soil, which has the most active organism–environment interaction, we found a well-constrained C:N ratio (14.4, molar ratio) and relatively consistent C:P (136) and N:P (9.3) ratios, with a general C:N:P ratio of 134:9:1. Finally, we suggested that soil C:N, C:P and N:P ratios in organic-rich topsoil could be a good indicator of soil nutrient status during soil development.     

Plant—Phytoseiid Interactions Mediated by Herbivore-Induced Plant Volatiles: Variation in Production of Cues and in Responses of Predatory Mites

Phytoseiid mites use herbivore-induced plant volatiles in long-range prey-habitat location and are arrested by these volatiles in a prey patch. The responses of predatory mites to these volatiles are considered to be an important factor in the local extermination of prey populations by phytoseiids such as Phytoseiulus persimilis. Prey-induced plant volatiles are highly detectable and can be reliable indicators of prey presence and prey identity. The composition of herbivore-induced plant volatiles depends on plant species and plant cultivar. Moreover, the composition may also vary with the herbivore species that infests a plant. The responses of phytoseiids to prey-induced plant volatiles from a specific plant-herbivore combination are highly variable. Causal factors include starvation, specific hunger, experience, pathogen infestation and the presence of competitors. Investigating variation in the phytoseiid's behavioural response in relation to these factors is important for understanding how and why behavioural strategies maximize phytoseiid fitness.