Abstract
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.
Highlights
Human chromosomes contain many guanine (G)-rich elements capable of forming four-stranded G-quadruplex (G4) structures (Bochman et al, 2012; Huppert and Balasubramanian, 2005; Maizels and Gray, 2013; Rhodes and Lipps, 2015)
Intermolecular G4 are substrates for telomerase, whereas intramolecular antiparallel or hybrid conformations are not (Hwang et al, 2014; Lee et al, 2017; Moye et al, 2015; Oganesian et al, 2006; Zahler et al, 1991; Zaug et al, 2005). Determining whether it is the parallel or intermolecular nature of G4 structures that allows their recognition by telomerase has been difficult, since a 4-repeat human telomeric oligonucleotide does not readily fold into stable parallel intramolecular G4 at the concentrations used in in vitro assays, and instead exists as a mixture of topologies under most conditions (Dai et al, 2007; Long and Stone, 2013; Palackyet al., 2013; Petraccone et al, 2012)
We introduced an aspartate-to-alanine mutation at highly conserved telomerase reverse transcriptase protein (hTERT) amino acid 712 and confirmed that this mutant telomerase (D712A) lost all primer extension activity (Figure 5— figure supplement 1). single-molecule fluorescence resonance energy transfer (smFRET) experiments with F-[7GGT]4 demonstrated an initial drop in FRET after addition of D712A telomerase, but no further drop in FRET was observed upon addition of deoxynucleotide triphosphates (dNTPs) (Figure 5A–E)
Summary
Human chromosomes contain many guanine (G)-rich elements capable of forming four-stranded G-quadruplex (G4) structures (Bochman et al, 2012; Huppert and Balasubramanian, 2005; Maizels and Gray, 2013; Rhodes and Lipps, 2015). We have demonstrated that telomerase can bind and extend specific conformations of telomeric G4 - those that are parallel-stranded and intermolecular - and that this property of telomerase is well conserved from ciliates to human (Moye et al, 2015; Oganesian et al, 2007; Oganesian et al, 2006). This suggests that if telomeric G-quadruplexes were to adopt a parallel conformation in vivo, they may be extended by telomerase, contrary to what had been previously hypothesized. We provide a mechanistic explanation of conformation-specific telomerase extension of telomeric G4 at singlemolecule resolution and demonstrate that small molecule-mediated inhibition of telomerase extension of telomeric G4 is topology-dependent
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