136 research outputs found
A one-step method for quantitative determination of uracil in DNA by real-time PCR
Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome
Crystallization and preliminary crystallographic analysis of dUTPase from the helper phage Φ11 of Staphylococcus aureus
Staphylococcus aureus superantigen-carrying pathogenicity islands (SaPIs) have a
determinant role in spreading virulence genes among bacterial populations that constitute a
major health hazard. Repressor (Stl) proteins are responsible for transcriptional regulation of
pathogenicity island genes. Recently, a derepressing interaction between the repressor Stl
SaPIbov1 with dUTPase from the Φ11 helper phage was suggested [Tormo-Mas et al. (2010).
Nature 465, 779-782]. Towards elucidating the molecular mechanism of this interaction, this
study reports expression, purification, and X-ray analysis of Φ11 dUTPase that contains a
phage-specific polypeptide segment not present in other dUTPases. Crystals were obtained
using the hanging-drop vapor-diffusion method at room temperature. Data were collected
from one type of crystal to 2.98 Å resolution. The crystal of Φ11 dUTPase belonged to the
cubic space group I23, with unit-cell parameters a=98.16 Å, α=β=γ= 90.00o
Factors influencing nucleo-cytoplasmic trafficking: which matter? Response to Alvisi & Jans' comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights
The authors respond to a comment by Alvisi & Jans [(2014), Acta Cryst. D70, 2775-2776] on the article Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights [Róna et al. (2013), Acta Cryst. D69, 2495-2505]
Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.
Lipid-protein interactions are rarely characterized
at a structural molecular level due to technical
difficulties; however, the biological significance of understanding the mechanism of these interactions is
outstanding. In this report, we provide mechanistic
insight into the inhibitory complex formation of the
lipid mediator sphingosylphosphorylcholine with calmodulin,
the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic,
thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the
calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well
Mechanism-Based Redesign of GAP to Activate Oncogenic Ras
Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously
Reducing data dimension boosts neural network-based stage-specific malaria detection
Although malaria has been known for more than 4 thousand years(1), it still imposes a global burden with approx. 240 million annual cases(2). Improvement in diagnostic techniques is a prerequisite for its global elimination. Despite its main limitations, being time-consuming and subjective, light microscopy on Giemsa-stained blood smears is still the gold-standard diagnostic method used worldwide. Autonomous computer assisted recognition of malaria infected red blood cells (RBCs) using neural networks (NNs) has the potential to overcome these deficiencies, if a fast, high-accuracy detection can be achieved using low computational power and limited sets of microscopy images for training the NN. Here, we report on a novel NN-based scheme that is capable of the high-speed classification of RBCs into four categories—healthy ones and three classes of infected ones according to the parasite age—with an accuracy as high as 98%. Importantly, we observe that a smart reduction of data dimension, using characteristic one-dimensional cross-sections of the RBC images, not only speeds up the classification but also significantly improves its performance with respect to the usual two-dimensional NN schemes. Via comparative studies on RBC images recorded by two additional techniques, fluorescence and atomic force microscopy, we demonstrate that our method is universally applicable for different types of microscopy images. This robustness against imaging platform-specific features is crucial for diagnostic applications. Our approach for the reduction of data dimension could be straightforwardly generalised for the classification of different parasites, cells and other types of objects
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