Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.
, J. L. (2015). ESI-MS investigation of an equilibrium between a bimolecular quadruplex DNA and a duplex DNA/RNA hybrid. Journal of the American Society for Mass Spectrometry, 26 (7), 1165-1173.ESI-MS investigation of an equilibrium between a bimolecular quadruplex DNA and a duplex DNA/RNA hybrid AbstractElectrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the quadruplex DNA (qDNA) resulted in formation of the duplex DNA/ RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its circular dichroism (CD) melting temperature of 10°C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid. Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if telomeric DNA was folded into a qDNA structure, it is possible for a DNA/RNA hybrid to form as is required during template alignment. The discrimination of ligand (1) AbstractElectrospray ionization mass spectrometry (ESI-MS) conditions were optimized for simultaneous observation of a bimolecular qDNA and a Watson-Crick base-paired duplex DNA/RNA hybrid. The DNA sequence used was telomeric DNA, and the RNA contained the template for telomerase-mediated telomeric DNA synthesis. Addition of RNA to the qDNA resulted in formation of the duplex DNA/RNA hybrid. Melting profiles obtained using circular dichroism spectroscopy confirmed that the DNA/RNA hybrid exhibited greater thermal stability than the bimolecular qDNA in solution. Binding of a 13-substituted berberine (1) derivative to the bimolecular qDNA stabilized its structure as evidenced by an increase in its stability in the mass spectrometer, and an increase in its (CD) melting temperature of 10 C. The DNA/RNA hybrid did not bind the ligand extensively and its thermal stability was unchanged in the presence of (1). The qDNA-ligand complex resisted unfolding in the presence of excess RNA, limiting the formation of the DNA/RNA hybrid.Previously, it has been proposed that DNA secondary structures, such as qDNA, may be involved in the telomerase mechanism. DNA/RNA hybrid structures occur at the active site of telomerase. The results presented in the current work show that if tel...
Two different series of nickel Schiff base complexes were prepared as part of a study aimed at discovering new compounds with high affinity and selectivity for quadruplex DNA (qDNA). The new complexes were prepared by modification of a literature method for synthesising N,N'-bis-(4-((1-(2-ethyl)piperidine)-oxy)salicylidene)phenylenediaminenickel(ii) (complex (1)). For Series 1 complexes, the phenylenediamine head group of the literature complex was replaced with ethylenediamine, phenanthrenediamine, R,R- and S,S-diaminocyclohexane. These complexes, as well as an asymmetric molecule featuring a naphthalene moiety on one side and a single ethyl piperidinyl salicylidene group on the other, were prepared in order to examine the effect of varying the number and position of aromatic groups on DNA binding. Series 2 complexes were isomers of those in Series 1, in which pendant ethyl piperidine groups were located at different positions. All new complexes were characterised by 1D and 2D NMR spectroscopic methods alongside microanalysis and ESI-MS. In addition, the solid state structures of eight new complexes were determined using single crystal X-ray diffraction methods. N,N'-Bis-(4-((1-(2-ethyl)piperidine)oxy)-salicylidine)diaminophenanthrenenickel(ii) (9), was shown by ESI-MS, CD spectroscopy and UV melting studies to exhibit a greater affinity towards, and ability to stabilise, dsDNA than all other complexes in the first series. ESI-MS revealed (9) to have a strong tendency to form a 1 : 1 complex with the tetramolecular, parallel qDNA molecule Q4, however it exhibited low affinity towards the parallel unimolecular qDNA molecule Q1. The enantiomeric complexes (5) and (7), featuring R,R- and S,S-diaminocyclohexane moieties, respectively, showed similar binding profiles towards all DNA molecules investigated, whereas the asymmetric complex (11), exhibited very low DNA affinity in all cases. Series 2 complexes showed very similar DNA affinity and selectivity to their isomeric counterparts in Series 1. For example, (14) and (15), both of which contain a phenylenediamine head group, showed higher affinity towards D2, Q1 and Q4, than any of the other Series 2 complexes. In addition, complex (21), which contains a meso-1,2-diphenylethylenediamine moiety, interacted strongly with Q4, but not D2 or Q1. This observation was very similar to that made previously for the isomeric complex (3).
1Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, 2 preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have 3 previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended 4 by human and ciliate telomerase. However, a mechanistic understanding of the interaction of 5 telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence 6 resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a 7 mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated 8 by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then 9 proceeds to the end of the RNA template. It is only through the large conformational change 10 of translocation following synthesis that the G-quadruplex structure is completely unfolded to 11 a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or 12 by chemical modification does not always inhibit G4 unfolding and extension by telomerase. 13These data reveal that telomerase is a parallel G-quadruplex resolvase.14 Human chromosomes contain many guanine (G)-rich elements capable of forming four-1 stranded G-quadruplex (G4) structures (1-4). A planar G-quartet is formed when four Gs form 2 hydrogen bonds in a cyclical manner via their Hoogsteen faces; such G-quartets stack on top 3 of each other and form G-quadruplexes (5, 6). G-rich sequences are primarily located in 4 promoter regions, intron and exon boundaries, origins of replication and telomeres (1-4). 5Vertebrate telomeres consist of many tandem repeats of the sequence TTAGGG, comprising a 6 double-stranded region and a 3' G-rich overhang (7). Fluorescence microscopy studies have 7 reached, telomerase translocates downstream, resulting in re-alignment of the RNA template 1 with the new 3' end of the product DNA (19, 22). 2One function of telomeric G4 may be to act as a 'cap', protecting the telomere from DNA 3 degradation (24). It has been widely believed that G4 formation within the 3' telomeric 4 overhang blocks telomere extension by telomerase, since early in vitro studies had shown that 5 G4 structures inhibit telomerase extension of a telomeric DNA substrate (25,26). In addition, 6 stabilization of G4 with small-molecule ligands has been shown to more effectively inhibit 7 telomerase activity, suggesting that chemical stabilization of G4 structures may be a viable 8 anti-cancer therapeutic strategy (27)(28)(29). However, the above-mentioned studies did not 9 distinguish between G4 conformations, and used oligonucleotides that likely folded into anti-10 parallel or 'hybrid' G4 forms as a telomerase substrate. A variety of helicases, such as RECQ5, 11 WRN, BLM, FANC-J and RHAU, recognize and resolve G4 in a conformation-specific 12 manner (30)(31)(32)(33). Similarly, we have demonstrated that telomerase can bind and extend specific 13 conformations of telomeric G4 -those t...
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