Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere ...junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA's annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Telomere resolvases are a family of DNA cleavage and rejoining enzymes that produce linear DNAs terminated by hairpin telomeres from replicated intermediates in bacteria that possess linear ...replicons. The telomere resolvase of Agrobacterium tumefaciens, TelA, has been examined at the structural and biochemical level. The N-terminal domain of TelA, while not required for telomere resolution, has been demonstrated to play an autoinhibitory role in telomere resolution, conferring divalent metal responsiveness on the reaction. The N-terminal domain also inhibits the competing reactions of hp telomere fusion and recombination between replicated telomere junctions. Due to the absence of the N-terminal domain from TelA/DNA co-crystal structures we produced an AlphaFold model of a TelA monomer. The AlphaFold model suggested the presence of two inhibitory interfaces; one between the N-terminal domain and the catalytic domain and a second interface between the C-terminal helix and the N-core domain of the protein. We produced mutant TelA's designed to weaken these putative interfaces to test the validity of the modeled interfaces. While our analysis did not bear out the details of the predicted interfaces the model was, nonetheless, extremely useful in guiding design of mutations that, when combined, demonstrated an additive activation of TelA exceeding 250-fold. For some of these hyperactive mutants stimulation of telomere resolution has also been accompanied by activation of competing reactions. However, we have also characterized hyperactive TelA mutants that retain enough autoinhibition to suppress the competing reactions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of Agrobacterium tumefaciens in a process referred to as telomere resolution. Telomere resolution is a unique DNA ...cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3'-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5'-OH ends of the refolded strands attack the 3'-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Fair Food Program (FFP) provides a mechanism through which agricultural workers’ collective voice is expressed, heard and responded to within global value chains. The FFP's model of worker‐driven ...social responsibility presents an alternative to traditional corporate social responsibility. This article identifies the FFP's key components and demonstrates its resilience by identifying the ways in which the issues faced by a new group of migrant workers – recruited through a “guest‐worker” scheme – were incorporated and dealt with. This case study highlights the important potential presented by the programme to address labour abuses across transnationalized labour markets while considering early replication possibilities.
Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is ...circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein–protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.
In spite of serving as the purported goal of the global production network (GPN) approach, development has been left undefined in the GPN literature, with ‘GPN 2.0’ now offering an impoverished ...understanding of development. This article reviews the elaboration of the ‘core concepts’ of the GPN approach: value, power, embeddedness – and development. I argue that the dis/articulations perspective is useful in offering a critical interrogation of d/Development, and that this has implications for value, power and embeddedness. The disarticulations perspective takes the determination of value into account and highlights the role of borders and discursive boundaries in structuring power relations
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DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exploitation of international migrant workers in the Global North has been increasingly framed in terms of trafficking, in political and legal domains and by the media. Yet posing trafficking as a ...phenomenon that captures the unfreedom experienced by migrants obscures the variegated means through which unfree labour relations are both institutionalized, and related to more ‘mundane’ forms of exploitation including precarious employment (for migrants and non-migrants alike). In this paper we argue that conceptualizing forms of unfreedom along a continuum of labour relations highlights this interrelationship, which for migrant workers includes attempts to harness and control mobilities through immigration regimes that restrict mobility bargaining power within labour markets. We use the example of the Temporary Foreign Worker Program (TFWP) in Canada to show how precarious employment, precarious legal status and unfree labour relations interact, and how they are negotiated and contested by of workers themselves.
RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study ...of recA mutation in other organisms. 'Missing' recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi's RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the 'missing' phenotypes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
► I examine the case of slave labour among migrant Brazilian sugar cane workers. ► I use the GPN framework to place unfree labour within production networks. ► I show how labour is de-valued within a ...GPN through processes of racialisation. ► I show how beyond institutional context, the state exercises governance within GPNs. ► I show how efforts to combat slave labour work through GPN power dynamics.
This article presents an analysis of slave labour (as it is known in Brazil) among sugar cane workers within a globalising production network. It employs the Global Production Network (GPN) framework to argue that the dynamics of production networks are fundamental to the reproduction of unfree and degrading labour in this case. First, the power exercised by buyers is a key aspect of processes resulting in slave labour. Conversely, efforts to combat slave labour have been strengthened by acknowledging and working through this power. Second, the state exercises governance within the production network rather than only providing its institutional context. Beyond these dynamics, however, wider processes are involved in making labour available on particular terms and conditions. Third, then, processes of racialisation facilitate the imposition of restrictions on workers’ mobility, degrading conditions and intensification of work. Labour is, in other words, devalued. This implies that the ways in which competing judgments over value are resolved merit as much attention in GPN analysis as is currently given to the creation, enhancement and capture of value.
Trafficking, forced labour and ‘slavery’ (TFLS) have become a central cause for our time, but anti-TFLS efforts have also come under forceful criticism. Amidst these ongoing debates, we observe that ...TFLS is currently being reframed as a problem of and for development. We consider the implications of this reframing by first reviewing the tangled history of abolitionism, colonialism and development, linking this to critical understandings of development more broadly. We then utilise Critical Discourse Analysis (CDA) to study the methodological assumptions and discursive framing of (anti-)TFLS in two paradigmatic texts. In doing so, we trace an important discursive shift – to anti-TFLS as development – in the moment it unfolds. Troubling the narrative of anti-TFLS as development, we conclude that while it promises to amend the criminal justice approach, it nevertheless perpetuates a global politics of rescue.