436 research outputs found

    The Impact of the C-Terminal Domain on the Interaction of Human DNA Topoisomerase II Ξ± and Ξ² with DNA

    Get PDF
    <b>Background</b> Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIΞ± and topoisomerase IIΞ², that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction. <b>Methodology/Principal Findings</b> We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIΞ± and topoisomerase IIΞ² to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIΞ² but not topoisomerase IIΞ± affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes. <b>Conclusions/Significance</b> The C-terminal domain of topoisomerase IIΞ², but not that of topoisomerase IIΞ±, alters the enzyme's KD for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIΞ± or topoisomerase IIΞ², either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn

    Collective Power to Create Political Change: Increasing the Political Efficacy and Engagement of Social Workers

    Get PDF
    Because social workers are called to challenge social injustices and create systemic change to support the well-being of individuals and communities, it is essential that social workers develop political efficacy: belief that the political system can work and they can influence the system. This study explored the impact of an intensive political social work curriculum on political efficacy and planned political engagement among social work students and practitioners. The findings suggest this model of delivering a political social work curriculum effectively increases internal, external, and overall political efficacy, and that increasing political efficacy has promise for increasing future political engagement

    Helical Chirality: a Link between Local Interactions and Global Topology in DNA

    Get PDF
    DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology

    A-Site Residues Move Independently from P-Site Residues in all-Atom Molecular Dynamics Simulations of the 70S Bacterial Ribosome

    Get PDF
    The ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and RNA chains. We ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. Because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. We hypothesize that residues that show coupled dynamics are functionally related, even on longer timescales. We validate our model by showing that crystallographic B-factors correlate well with the entropy calculated as part of our mutual information calculations. We reveal that A-site residues move relatively independently from P-site residues, effectively insulating A-site functions from P-site functions during translation

    T-Analyst: a program for efficient analysis of protein conformational changes by torsion angles

    Get PDF
    T-Analyst is a user-friendly computer program for analyzing trajectories from molecular modeling. Instead of using Cartesian coordinates for protein conformational analysis, T-Analyst is based on internal bond-angle-torsion coordinates in which internal torsion angle movements, such as side-chain rotations, can be easily detected. The program computes entropy and automatically detects and corrects angle periodicity to produce accurate rotameric states of dihedrals. It also clusters multiple conformations and detects dihedral rotations that contribute hinge-like motions. Correlated motions between selected dihedrals can also be observed from the correlation map. T-Analyst focuses on showing changes in protein flexibility between different states and selecting representative protein conformations for molecular docking studies. The program is provided with instructions and full source code in Perl

    Race discrimination, the politics of knowledge and cultural inequality in England

    Get PDF
    From the outside, one might assume England’s education system is tolerant of diversity, welcoming and pluralistic. The truth is, to some extent it is. There are thousands of Overseas Trained Teachers (OTTs) and teachers of Black, Asian and Minority Ethnic (BAME) heritage working in schools in England. These two groups of teachers experience one thing in common - race discrimination, built on assumptions of cultural inequality, fuelled by structures that promote a deficit model of difference and a politics of knowledge. For, although thousands of teachers work in English schools, only few from BAME heritage and migrant origin make it to leadership positions, despite having similar aspirations, qualifications and work experience. Drawing on evidence from research on OTTs and teachers of BAME heritage, this chapter will show that five items present as barriers to the progression of OTTs of BAME heritage and teachers of BAME heritage as a whole including: policy, race/racism, institutional practices, group membership and religion (Islam). This chapter also provides a reconceptualisation of promotion, progression for OTTs and teachers of BAME heritage based on three criteria: β€œAffiliation”, β€œAppeasement” (or β€œAdaptation”) and β€œWhite sanction”. The chapter considers epistemological and ontological misunderstandings of difference, and highlights the need for courageous and transformative leadership from policy makers and institutions in tackling race inequality in education in England

    Topoisomerase IIΞ± Binding Domains of Adenomatous Polyposis Coli Influence Cell Cycle Progression and Aneuploidy

    Get PDF
    Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind beta-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15-amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase IIalpha (topo IIalpha).We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIalpha in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIalpha isoform and lacking topo IIbeta did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIalpha. Of note, although M3-APC can bind beta-catenin, the G2 arrest did not correlate with beta-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region.Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIalpha to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC

    The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

    Get PDF
    Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs
    • …
    corecore