Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation

Abstract

<jats:title>SUMMARY</jats:title><jats:p>Cells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In <jats:italic>Bacillus subtilis</jats:italic>, the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters (<jats:italic>ori/ter</jats:italic> ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. <jats:italic>In vitro</jats:italic>, PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to <jats:italic>pykA</jats:italic>. Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.</jats:p>

Which training leads to employment? The effectiveness of varying types of training programmes for unemployed jobseekers in Flanders

“…Two important unaddressed theoretical barriers to employment are low human capital and productivity (requiring more advance training), and limited opportunities to convince potential employers (requiring workplace training). This conclusion aligns with contemporary policy evolutions in Flanders to consider additional components to be included in general classroom training or combine such training with follow-up programmes (Horemans & Ghysels, 2020).…”

“…Consequently, policymakers might reconsider whether the employment office can act as a matchmaker between jobseekers and employers willing to participate in contractual workplace training, rather than leaving the initiative to employers who who are prone to cream-skimming. This recommendation aligns with recent evolutions in Flanders strengthening the employment office's activities around contractual workplace training (Horemans & Ghysels, 2020;Vlaamse Regering, 2014. Contributing to an inconclusive body of evaluations of workplace experience programmes and temporary wage subsidies (Card et al, 2017;Card et al, 2010;Dahl & Lorentzen, 2005;Gerfin & Lechner, 2002), our interpretation of the limited effectiveness of contractual workplace training in terms of selective recruitment resonates with long-standing findings of deadweight losses of wage subsidies in case participants would have also been hired in the absence of wage subsidies (Bell et al, 1999;Marx, 2001).…”