A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

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.

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…Arginine and lysine interacted with nucleic acids in a manner similar to monovalent cations, and arginine had strong chirality dependence on the inhibition of TL-TLR folding [102,103] TMAO and urea were demonstrated to alter the nucleic acid folding by osmotic pressure [104] Chromosome Structure Strand Sequences PNA-probe binding was detected repeatedly to evaluate the accessibility in the telomere area [109] Presence of telomerase Enzymatic destruction of telomere was described and computer simulation was coupled [110,111] Solvent condition Primer-labeling method used to determine the dynamics of single nucleosomes [114][115][116][117] DNA damage Chromatin function in DNA damage response was observed dynamically in living cells [89,120]…”

“…smFRET exhibited not only the structural features but also the process of destruction of telomeres [ 110 ]. Fluorophores were labeled at each side of G4 structures in pairs to monitor the disruption of parallel G4 by telomerase translocation.…”

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [39][40][41][42], DNA combing and curtains [43][44][45][46], DNA tightropes (via oblique angle fluorescence imaging) [47,48], as well as smFRET [40,[49][50][51][52][53] and TIRF/AFM systems [54][55][56]. While the examples mentioned below include data collected on both prism and objective-based TIRF instruments, in most cases, the prismTIRF microscope described above can be used for similarly designed experiments.…”

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [36][37][38][39], DNA combing and curtains [40][41][42][43], DNA tightropes (via oblique angle fluorescence imaging) [44,45], as well as smFRET [37,[46][47][48][49][50] and TIRF/AFM systems [51][52][53]. While the examples mentioned below include data collected on both prism and objective based TIRF instruments, in most cases the prismTIRF microscope described above can be used for similarly designed experiments.…”