Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3') n , as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT). This repeat is of particular interest because it contains a run of three contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ∼12-fold. The bead-like structures were 5 and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ∼28 nm at forces between 6 and 12 pN, well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed.
Keywords: DNA; G-quadruplex; electron microscopy (EM); single-molecule biophysics; telomere.
© 2018 Kar et al.