Mechanistic insights into type III restriction enzymes

Type III restriction-modification (R-M) enzymes need to interact with two separate unmethylated DNA sequences in indirectly repeated, head-to-head orientations for efficient cleavage to occur at a defined location next to only one of the two sites. However, cleavage of sites that are not in head-to-head orientation have been observed to occur under certain reaction conditions in vitro. ATP hydrolysis is required for the long-distance communication between the sites prior to cleavage. Type III R-M enzymes comprise two subunits, Res and Mod that form a homodimeric Mod(2) and a heterotetrameric Res(2)Mod(2) complex. The Mod subunit in M-2 or R2M2 complex recognizes and methylates DNA while the Res subunit in R2M2 complex is responsible for ATP hydrolysis, DNA translocation and cleavage. A vast majority of biochemical studies on Type III R-M enzymes have been undertaken using two closely related enzymes, EcoP1I and EcoP15I. Divergent opinions about how the long-distance interaction between the recognition sites exist and at least three mechanistic models based on 1D- diffusion and/or 3D-DNA looping have been proposed.

Novel algorithm for online voltage stability assessment based on feed forward neural network

This paper presents an artificial feed forward neural network (FFNN) approach for the assessment of power system voltage stability. A novel approach based on the input-output relation between real and reactive power, as well as voltage vectors for generators and load buses is used to train the neural net (NN). The input properties of the feed forward network are generated from offline training data with various simulated loading conditions using a conventional voltage stability algorithm based on the L-index. The neural network is trained for the L-index output as the target vector for each of the system loads. Two separate trained NN, corresponding to normal loading and contingency, are investigated on the 367 node practical power system network. The performance of the trained artificial neural network (ANN) is also investigated on the system under various voltage stability assessment conditions. As compared to the computationally intensive benchmark conventional software, near accurate results in the value of L-index and thus the voltage profile were obtained. Proposed algorithm is fast, robust and accurate and can be used online for predicting the L-indices of all the power system buses. The proposed ANN approach is also shown to be effective and computationally feasible in voltage stability assessment as well as potential enhancements within an overall energy management system in order to determining local and global stability indices

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

Most bacterial genomes harbor restriction-modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host-parasite coevolutionary ``arms race.''

Influence of tool rotation speed and feed rate on the forming limit of friction stir welded AA6061-T6 sheets

In this study, the influence of tool rotation speed and feed rate on the forming limit of friction stir welded Al 6061-T651 sheets has been investigated. The forming limit curve was evaluated by limit dome height test performed on all the friction stir welded sheets. The welding trials were conducted at a tool rotation speed of 1300 and 1400 r/min and feed rate of 90 and 100 mm/min. A third trial of welding was performed at a rotational speed of 1500 r/min and feed rate 120 mm/min. It is found that with increase in the tool rotation speed, from 1300 to 1400 r/min, for a constant feed rate, the forming limit of friction stir welded blank has improved and with increase in feed rate, from 90 to 100 mm/min, for a constant tool rotation speed, it has decreased. The forming limit of friction stir welded sheets is better than unwelded sheets. The thickness gradient after forming is severe in the cases of friction stir welded blanks made at higher feed rate and lower rotation speed. The strain hardening exponent of weld (n) increases with increase in tool rotation speed and it decreases with increase in feed rate. It has been demonstrated that the change in the forming limit of friction stir welded sheets with respect to welding parameters is due to the thickness distribution severity and strain hardening exponent of the weld region during forming. There is not much variation in the dome height among the friction stir welded sheets tested. When compared with unwelded sheets, dome height of friction stir welded sheets is higher in near-plane-strain condition, but it is lesser in stretching strain paths.

Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exo-nucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori.

Diverse functions of restriction-modification systems in addition to cellular defense

Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 mu M mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

The influence of termites on soil sheeting properties varies depending on the materials on which they feed

Fungus-growing termites are involved in many ecological processes and play a central role in influencing soil dynamics in the tropics. The physical and chemical properties of their nest structures have been largely described; however less information is available concerning the relatively temporary structures made above-ground to access food items and protect the foraging space (the soil `sheetings'). This study investigated whether the soil physical and chemical properties of these constructions are constant or if they vary depending on the type of food they cover. Soil samples and soil sheetings were collected in a forest in India, from leaves on the ground (LEAF), fallen branches (WOOD), and vertical soil sheetings covering the bark of trees (TREE). In this environment, termite diversity was dominated by Odontotermes species, and especially Odontotermes feae and Odontotermes obesus. However, there was no clear niche differentiation and, for example, O. feae termites were found on all the materials. Compared with the putative parent soil (control), TREE sheetings showed the greatest (and most significant) differences (higher clay content and smaller clay particle sizes, lower C and N content and smaller delta C-13 and delta N-15), while LEAF sheetings were the least modified, though still significantly different than the control soil. We suggest that the termite diversity is a less important driver of potential soil modification than sheeting diversity. Further, there is evidence that construction properties are adapted to their prospective life-span, with relatively long-lasting structures being most different from the parent soil. (C) 2015 Elsevier Masson SAS. All rights reserved.

The Object Agreement Restriction

[EN]This paper deals with the so-called Person Case Constraint (Bonet, 1991), a universal constraint blocking accusative clitics and object agreement morphemes other than third person when a dative is inserted in the same clitic/agreement cluster. The aim of this paper is twofold. First, we argue that the scope of the PCC is considerably broader than assumed in previous work, and that neither its formulation in terms of person (1st/2nd vs. 3rd)-case (accusative vs. dative) restrictions nor its morphological nature are part of the right descriptive generalization.We present evidence (i) that the PCC is triggered by the presence of an animacy feature in the object’s agreement set; (ii) that it is not case dependent, also showing up in languages that lack dative case; and (iii) that it is not morphologically bound. Second, we argue that the PCC, even if it is modified accordingly, still puts together two different properties of the agreement system that should be set apart: (i) a cross linguistic sensitivity of object agreement to animacy and (ii) a similarly widespread restriction on multiple object agreement observed crosslinguistically. These properties lead us to propose a new generalization, the Object Agreement Constraint (OAC): if the verbal complex encodes object agreement, no other argument can be licensed through verbal agreement.