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Adventures with Four-Stranded DNA structures
DNA can adopt many different conformations and amongst the best-studied are G-quadruplexes, four-stranded secondary structures formed from sequences rich in guanine.1 The regions of DNA which form in the cytosine-rich sequences opposite G-quadruplexes are also able to form i-motifs, also four stranded structures comprised of parallel-stranded DNA duplexes connected in an anti-parallel orientation by intercalated, cytosine–cytosine base pairs.2 i-Motifs are stabilised by acidic conditions, which promote C-C base pairing via hemi-protonation of the N3; folding is rapid and this property has been utilised in many different nanotechnological applications. It is often assumed that i-motif formation is dependent on low pH, but there have always been examples where they are still present under neutral conditions.3 There are also now cases where i-motif can form at neutral and slightly alkaline pH depending on the types of sequence,4,5 conditions6-8 and the presence of ligands.9 Moreover, there are examples of stabilisation of i-motif structures resulting in disruption of telomerase10 and enhancement of gene expression.11
Here we describe work in our group towards the stabilisation of four-stranded DNA structures and their applications in nanotechnology and biology.
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Biosynthesis of Nitrous Oxide Reductase: A Complex Bacterial Multicopper Enzyme Essential for the Global Nitrogen Cycle
Nitrous oxide (N2O) or ‘laughing gas’ is a very potent greenhouse gas produced as an intermediate metabolite during anaerobic respiration of nitrate by denitrifying soil microorganisms1,2. Nitrous oxide reductase (NosZ) is the enzyme responsible for the reduction of N2O to dinitrogen (N2) and its activity is highly dependent on copper (Cu)3. NosZ is a functional dimer and each monomer contains six Cu atoms distributed into two different redox centres: the catalytic site CuZ and the electron transfer site CuA.
Biosynthesis requires the action of Cu chaperones that bind Cu and assist during the metalation of the active NosZ holoenzyme. Some chaperones are encoded in the nos gene cluster and are thought to be responsible for assembly of the CuZ centre. However, our group has recently reported previously uncharacterised genes, in the model organism Paracoccus denitrificans, whose expression is modulated by extracellular copper availability3 and may be required for the maturation of the CuA centre of terminal reductases.
Here we report that disruption of pcuC and scoB leads to the accumulation of N2O by denitrifying bacterial cells. The biochemical properties of PcuC and ScoB have been determined and we demonstrate they are required to deliver Cu to N2O and perhaps other O2 dependent terminal reductases.
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4) Sullivan, M. J., Gates, A. J., Appia-Ayme, C., Rowley, G., Richardson, D. J. Proc. Natl. Acad. Sci. USA 2013, 110, 19926.