Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.     

Machine Learning of Hierarchical Clustering to Segment 2D and 3D Images

We aim to improve segmentation through the use of machine learning tools during region agglomeration. We propose an active learning approach for performing hierarchical agglomerative segmentation from superpixels. Our method combines multiple features at all scales of the agglomerative process, works for data with an arbitrary number of dimensions, and scales to very large datasets. We advocate the use of variation of information to measure segmentation accuracy, particularly in 3D electron microscopy (EM) images of neural tissue, and using this metric demonstrate an improvement over competing algorithms in EM and natural images.     

Estimation of the diversity between DNA calorimetric profiles,differential melting curves and corresponding melting temperatures

The Poland–Fixman–Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (H_GC?H_AT) between the helix‐coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with H_GC?H_AT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed H_GC?H_AT, the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (T_cal?T_m) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (T_cal?T_m) enlarge with the temperature melting range of the helix‐coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.