Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA).

NAD+-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD+-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo

Liberal peace/ethno-theocratic war: A biopolitical perspective on western policy in the Eelam war

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.This thesis develops a biopolitical perspective on Western states’ longstanding opposition to the formation of a Tamil state (Tamil Eelam) in the northeastern parts of the island of Sri Lanka (Ceylon). It does so by adopting and applying the concept of biopolitics as developed by Michel Foucault in the 1970s. Foucault used the idea of biopolitics to explain power relations and to consider peace through the matrix of war. He was especially interested in using this to understand power relations that emerged in the eighteenth century and especially in terms of the tensions between military confrontation and commercial expansion. This thesis adopts and applies the idea of biopolitics to the concept of liberal peace and its core principle, the security of global commerce, to offer a new interpretation of the rationale behind the opposition of Western states to the Tamil demand for political independence and their collaboration in Sri Lanka’s biopolitical transformation of the island into a Sinhala-Buddhist ethno-theocracy. As practitioners of the biopolitics of liberal peace, Western states have waged wars and collaborated in the wars of their Southern counterparts, allowing populations, including liberalised ones, to be killed, condoning the subversion of civil liberties, human rights and other democratic freedoms, including the right to selfdetermination of nations, that they simultaneously promote. The thesis explores the extent to which the collaboration of the West with the Sri Lankan state’s racist policies and counterinsurgency efforts is a continuation of the colonial policies of the British Empire in Ceylon. In developing a biopolitical perspective on the liberal state-building practices of the British Empire in colonial Ceylon, Sri Lanka’s adoption of the same practices, and the West’s own efforts to neutralise the Tamils’ armed struggle, the thesis explores the ways that power relations produce the effects of battle, and thus the way that peace becomes a means of waging war. When the power relations of law, finance, politics, and diplomacy produce the effects of battle, they become ways of waging war by other means. As well as being a thesis on Western policy in the war in Sri Lanka, the work is therefore also to some extent an attempt to see how far Foucault’s work on biopolitics might be pushed and developed and thus, at the same time, an attempt to turn the Foucauldian focus to an area thus far unexplored by those who have sought to engage with Foucault’s work

Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4

The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 °C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 °C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'

Micropropagation And The Incorporation Of Cyanobacterial Extracts On The Regeneration Of Ficus Carica Cv. Golden Orphan

Ficus carica L. (Moraceae) is a nutritious fruit rich in natural fibre and vitamins. The yellow fruited Golden Orphan cultivar is sweet and firm, suitable for commercialisation in Malaysia. However, conventional methods for plant propagation are less efficient, impeding commercial farming in Malaysia. The incorporation of cyanobacteria extracts in fig micropropagation has not been explored, yet alone with the nontoxic strains belonging from the genus Nostoc and Anabaena. This research aims to establish methods for the micropropagation of Golden Orphan using different plant growth regulators and cyanobacteria extracts. In vitro nodal bud explants were established in MS media with 1 mg/L BAP and induced shoots were treated with different concentrations and combinations of cytokinin (BAP, Kinetin, Thidiazuron and Zeatin) and auxins (NAA, IAA and IBA) for shoot multiplication. The induced shoots were also tested with different cyanobacteria strains namely Nostoc sp. Penang Hill (strain 1), Nostoc Antarctic (strain 2) and Anabaena sp. Penang Hill (strain 3) to evaluate explant morphogenic responses. Regenerated shoots were rooted in MS medium supplemented with auxins (NAA, IAA and IBA) and were acclimatised

Characterization of Agrobacterium tumefaciens DNA ligases C and D

Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Background: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 59 or 29 OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 59 phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2.A1.A3.A4.A0.A6.A8.A10 and T2.T3.T4.T6<T1.T8.T10. Bridging T’s generally gave higher yield of ligated product than bridging A’s. ATP concentrations above 33 mM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (.90%) joined a correctly paired, or T:G wobble-paired, substrate on the 39 side of the ligation junction while discriminating approximately 100-fold against most mispaire

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3′ side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5′-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV