Coupling Sequence-specific Recognition to DNA Modification

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is ∼28 Å away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.Sequence-specific modification of DNA is essential for nearly all forms of life and contributes to a myriad of biological processes including gene regulation, mismatch repair, host defense, DNA replication, and genetic imprinting. Methylation of cytosine and adenine bases is a key epigenetic process whereby phenotypic changes are inherited without altering the DNA sequence (1). The central role of the bacterial and mammalian S-adenosylmethionine (AdoMet)²-dependent DNA methyltransferases in virulence regulation and tumorigenesis, respectively, have led these enzymes to be validated targets for antibiotic and cancer therapies (2, 3). However, AdoMet-dependent enzymes catalyze diverse reactions, and the design of potent and selective DNA methyltransferase inhibitors is particularly challenging (4, 5). The design of drugs that bind outside the active site is a particularly attractive means of inhibition for enzymes with common cofactors like AdoMet because off-target inhibition often leads to toxicity (6). Unfortunately, robust methods to identify and characterize such critical binding sites distal from the active site have not been developed.DNA methyltransferases bind to a particular DNA sequence, stabilize the target base into an extrahelical position within the enzyme active site, and transfer the methyl moiety from AdoMet to the DNA (7). During this process, dramatic changes in the DNA structure such as bending, base flipping, or the intercalation of residues into the recognition sequence are often accompanied by large scale protein rearrangements (8). Here we characterized a specific conformational rearrangement of M.HhaI, a model DNA cytosine C⁵ methyltransferase with a cognate recognition sequence of 5′-GCGC-3′. Many structures of M.HhaI are available at high resolution including an ensemble of complexes with either cognate or nonspecific DNA (9, 10). Reorganization of an essential catalytic loop (residues 80–100) is regulated by sequence-specific protein-DNA interactions that occur ∼28 Å away from the catalytic loop (Fig. 1). Our work quantifies the importance of such distal communication in sequence-specific DNA modification and provides plausible structural mechanisms.Open in a separate windowFIGURE 1.Loop interactions in M.HhaI. A, two superimposed structures of M.HhaI are shown with the catalytic loop highlighted. Enzymes are in blue (open conformer) and red (closed conformer) with the cofactor in orange, the flipped cytosine in green, and position 1 of the DNA recognition sequence colored by atom along with Cys-81, Ile-86, and Arg-240. The large blue arrow shows the long-range structural communication between Arg-240 and the catalytic loop. B, close-up of the interactions between Arg-240, Ile-86, and position 1 of the recognition sequence. The flipped cytosine is in green. Removal of N² from the guanosine with an inosine base maintains near cognate loop motion while removal of O⁶ with a 2AP base has almost no loop motion.DNA-dependent positioning of the catalytic loop in M.HhaI was first observed crystallographically; cognate DNA stabilizes the loop-closed conformer, while nonspecific DNA leaves the loop in the open conformer (9, 10). Correct positioning of this loop is essential for catalysis because C81, the active site nucleophile that attacks the target cytosine base at the C⁶ position (supplemental Fig. S1), is ∼9.6 Å away in the loop-open conformer (Fig. 1A). Populating the closed conformer of the loop is essential for tight DNA binding and stabilizing the target cytosine that is flipped out of the DNA duplex (11–13). Using stopped-flow fluorescence spectroscopy to monitor the environment of tryptophan (Trp) residues inserted into the catalytic loop, we recently observed reorganization of this loop upon DNA binding in the absence of cofactor using the M.HhaI mutants W41F, W41F/K91W, and W41F/E94W (12). Loop positioning and the interconversion between the open and closed conformers, as determined from the intensities and rates of change in fluorescence signal are highly dependent on DNA sequence and confirm that cognate DNA stabilizes the loop-closed conformer whereas nonspecific DNA stabilizes the open conformer.In this study, W41F/K91W and W41F/E94W M.HhaI were preincubated with cognate (COG), non-cognate (NC), or nonspecific (NS) DNA and mixed with cofactor or cofactor product, AdoMet and S-adenosylhomocysteine (AdoHcy), respectively, in a stopped-flow apparatus. Differences in observed fluorescence intensity are indicative of shifts in the populations of the various loop conformers; no observable fluorescence change suggests no significant change in population and thus, essentially no loop positioning to the closed conformer. Non-cognate and cognate sequences are nearly identical but differ by a single base change, whereas the nonspecific DNA substrate has no similarity to the cognate sequence. As a methyltransferase searches for the cognate site within a genome, it must encounter both non-cognate and nonspecific DNA sequences and be able to distinguish these from the cognate sequence. We examine both binding, using the cofactor product AdoHcy, and catalysis, using the AdoMet cofactor of M.HhaI, with these various DNA substrates.     

Evolutionary dynamics of mating system shifts in Arabidopsis lyrata

Outcrossing is the prevalent mode of reproduction in plants and animals despite its substantial costs, while selfing and mixed mating occur at much lower frequency. Comparative research on plants has demonstrated the lability of self‐incompatibility, but there is little information about the transition on a within‐species level from self‐incompatibility to predominant selfing. We studied variation in mating system among 18 populations of Arabidopsis lyrata within a phylogenetic context to shed light on the evolution of selfing. Realized and potential mating systems were assessed by genetic analysis with microsatellite markers and hand‐self‐pollinations on 30 plants from each population. The fraction of self‐incompatible plants in a population was highly correlated with the outcrossing rate, showing that the spread of self‐compatibility is accompanied by or soon followed by an increase in the rate of selfing. The four predominantly selfing populations (outcrossing rates < 0.25) fell into more than one phylogenetic cluster, suggesting that the transition to selfing occurred more than once independently. Hence, A. lyrata offers an opportunity for the comparative analysis of outcrossing as a predominant mode of reproduction in plants and of the causes of the shift to selfing.     

The antimicrobial activity of compounds from the leaf and stem of Vitis amurensis against two oral pathogens

Nine compounds isolated from the leaf and stem of Vitis amurensis Rupr. (Vitaceae) were evaluated for their antimicrobial activity against two oral pathogens, Streptococcus mutans and Streptococcus sanguis, which are associated with caries and periodontal disease, respectively. The results of several antimicrobial tests, including MIC, MBC, and TBAI, showed that three compounds inhibited the growth of the test bacteria at concentrations ranging from 12.5 to 50 μg/mL. Among these compounds, compound 5, trans-ε-viniferin, displayed the strongest activity against S. mutans and S. sanguis with MIC values of 25 and 12.5 μg/mL, respectively. This is the first report on the antimicrobial activity of stilbenes and oligostilbenes isolated from the leaf and stem of V. amurensis. Thus, this result suggests that natural antimicrobial compounds derived from V. amurensis may benefit oral health as plaque-control agents for the prevention of dental caries and periodontal disease.     

1H-NMR study of the metabolome of a moderately hypoxia-tolerant fish, the common carp (Cyprinus carpio)

All 20.000 different fish species vary greatly in their ability to tolerate and survive fluctuating oxygen concentrations in the water. Especially fish of the genus Carassius, e.g. the crucian carp and the goldfish, exhibit a remarkable tolerance to limited/absent oxygen concentrations. The metabolic changes of anoxia-tolerant crucian carp were recently studied and published. Contrary to crucian carp, the hypoxia-tolerant common carp cannot survive a complete lack of oxygen (anoxia). Therefore, we studied the ¹H-NMR-based metabolomics of brain, heart, liver and white muscle extracts of common carp, subjected to anoxia (0 mg O₂ l^?1) and hypoxia (0.9 mg O₂ l^?1) at 5 °C. Specifically, fish were exposed to normoxia (i.e. 9 mg O₂ l^?1; controls 24 h, 1 week and 2 weeks), acute hypoxia (24 h), chronic hypoxia (1 week) and chronic hypoxia (1 week) with normoxic reoxygenation (1 week). Additionally, we also investigated the metabolic responses of fish to anoxia for 2 h. Both anoxia and hypoxia significantly changed the tissue levels of standard energy metabolites as lactate, glycogen, ATP/ADP and phosphocreatine. Remarkably, anoxia induced increased lactate levels in all tissues except for the heart whereas hypoxia resulted in decreased lactate concentrations in all tissues except for brains. Furthermore, hypoxia and anoxia influenced amino acids (alanine, valine/(iso)leucine) and neurotransmitters levels (GABA, glutamate). Lastly, we also detected ‘other’ i.e. previously not reported compounds to play a role in the present context. Scyllo-inositol levels changed significantly in heart, liver and muscle, providing novel insights into the anoxia/hypoxic responses of the common carp.     

Two-and Three-Dimensional Simulations of Beetle Hind Wing Flapping during Free Forward Flight

Aerodynamic characteristic of the beetle, Trypoxylus dichotomus, which has a pair of elytra (forewings) and hind wings, is numerically investigated. Based on the experimental results of wing kinematics, two-dimensional (2D) and three-dimensional (3D) computational fluid dynamic simulations were carried out to reveal aerodynamic performance of the hind wing. The roles of the spiral Leading Edge Vortex (LEV) and the spanwise flow were clarified by comparing 2D and 3D simulations. Mainly due to pitching down of chord line during downstroke in highly inclined stroke plane, relatively high averaged thrust was produced in the free forward flight of the beetle. The effects of the local corrugation and the camber variation were also investigated for the beetle's hind wings. Our results show that the camber variation plays a significant role in improving both lift and thrust in the flapping. On the other hand, the local corrugation pattern has no significant effect on the aerodynamic force due to large angle of attack during flapping.     

Nuclear cyp73 intron fragment length polymorphism supports morphological analysis of Salix species and hybrids

Abstract

The polyploid Salix alba L.–Salix fragilis L. hybrid complex still presents major difficulties in morphological identification. Most of the measured characters show a low diagnostic value for unambiguously identifying the parental species and their hybrid Salix × rubens Schrank due to continuous variation creating a large overlap in leaf and catkin morphology. Fragment length polymorphism of nuclear cyp73 intron markers was used to identify species and hybrids. This multilocus genotyping could be applied in a morphological analysis of trees from hybrid zones and allowed to demonstrate that morphological features of leaves and catkins clearly separated S. alba from S. fragilis. The hybrid individuals largely overlapped with both parental species but appeared to be morphologically more similar to S. fragilis than to S. alba. Cyp73 analysis of 11 Salix taxa revealed intermediate positions of two hybrid taxa with S. alba, namely S. × rubens and S. × sepulcralis Simonkai with their respective parental species S. fragilis and S. babylonica L. Additionally, the cyp73 intron multilocus genotypes clustered tetraploid taxa separately from diploid willows. Cyp73 introns are valuable markers for fast, reliable and straightforward genotyping in willow species and hybrids.     

Do hydration dynamics follow the structural perturbation during thermal denaturation of a protein: a terahertz absorption study

We investigate the thermal denaturation of human serum albumin and the associated solvation using terahertz (THz) spectroscopy in aqueous buffer solution. Far- and near-ultraviolet circular dichroism spectroscopy reveal that the protein undergoes a native (N) to extended (E) state transition at temperature ≤55°C with a marginal change in the secondary and tertiary structure. At 70°C, the protein transforms into an unfolded (U) state with significant irreversible disruption of its structures. We measure the concentration- and temperature-dependent THz absorption coefficient (α) of the protein solution using a p-Ge THz difference spectrometer (2.1–2.8 THz frequency range), thereby probing the collective protein-water network dynamics. When the solvated protein is heated up to 55°C and cooled down again, a reversible change in THz absorption is observed. When increasing the temperature up to 70°C, we find a dramatic irreversible change of THz absorption. The increase in THz absorption compared to bulk water is attributed to a blue shift in the spectrum of the solvated protein compared to bulk water. This is supported by measurements of THz absorption coefficients using THz time-domain spectroscopy (0.1–1.2 THz frequency range). We also use picosecond-resolved fluorescence spectroscopy of the tryptophan 214 moiety of human serum albumin. All experimental observations can be explained by a change in the hydration dynamics of the solvated protein due to the additional exposure of hydrophobic residues upon unfolding.     

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.