Mechanical Properties and Strand Invasion of Duplex Telomere DNA Probed using Magnetic Tweezers

Abstract

Telomeres are specialized chromatin structures that protect chromosome ends from nucleolytic processing by DNA repair machinery. The foundation of human telomere structure consists of a long array of tandem duplex DNA sequences (TTAGGG) and terminates with a single-stranded 3’ end. To protect the chromosome end, telomeres are thought to adopt a lariat structure known as a telomere-loop (T-loop)1. T-loops are stabilized by DNA displacement loops (D-loops) generated by the invasion of a single-stranded telomeric DNA tail into an adjacent region of duplex telomere. Recent studies suggest that telomere-associated proteins promote strand invasion during telomeric D-loop formation through the application of torque to the DNA2. Although the molecular mechanism of T-loop formation has been described using biochemical approaches, the torque response and internal structural equilibrium of duplex telomeric DNA are not well characterized. To probe the mechanical properties of duplex telomeric DNA, we developed a magnetic tweezers assay to detect the response of single telomeric DNA molecules to precisely applied degrees of tension and torque. Rotation-extension curves under varying tension demonstrate that the repetitive telomere DNA sequence is more refractory to torque-induced denaturation than a non-telomeric control molecule of comparable GC content. In addition, force-extension analysis of negatively supercoiled telomeric DNA in the presence of different counter-ions (K+ vs. Li+), reveals that transient torque-induced denaturation of duplex telomeric DNA promotes a structural transition into stable DNA G-quadruplexes. Lastly, using a single molecule DNA topology-based assay, we directly monitor the torque-dependent invasion of single stranded telomere DNA primers into duplex telomeric DNA tethers. Our results provide insight into the molecular mechanisms of telomere-associated proteins and enzymes during structural remodeling of telomeres.1. Griffith JD, et al. Cell 97: 503-514.2. Amiard S, et al. Nat Struct Mol Biol 14:147-154.