Telomeres are specialized nucleoprotein structures that protect the ends of linear chromosomes. In human cells, telomeres harbor 100-200 nt long tandem repeats of T2AG3 at the 3’ end. Although structural studies have demonstrated G-Quadruplex (GQ) formation in telomeres, their conformational dynamics under physiological levels of tension is poorly understood. We integrated the unique capabilities of optical tweezers and fluorescence resonance energy transfer to canvas the mechanical response of telomeric GQs, under near physiological conditions. In a telomeric sequence spanning four T2AG3 repeats, we observed at least six distinct, interconvertible species that differ in mechanical responses, but are largely resistant to forces exerted by cellular motor proteins. Refolding of GQs was rapid and occurred at much lower forces of ~ 2 pN. Truncation of the above sequence to three hexanucleotide repeats resulted in single-stranded DNA like behavior, highlighting that at least four repeats are essential for formation of mechanically stable secondary structures. We further demonstrated preferential GQ formation at the 3’ end of sequences containing five and six T2AG3 repeats, using a superhelicase-assisted vectorial folding assay. However, the relative position of the unassociated T2AG3 motif regulates the distribution of GQ species into mechanically distinct states. Location and conformational dynamics of GQ on a long G-rich telomeric overhang has important implications in processes such as telomerase inhibition, alternative lengthening of telomeres, T-loop formation, telomere end protection and replication.

Extreme mechanical diversity of human telomeric DNA probed by single-molecule fluorescence-force spectroscopy

2019 Fall National Meeting

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