Long repeating (TTAGGG)n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation

Anirban Kar,Nathan Jones,N Özlem Arat,Richard Fishel,Jack D Griffith

doi:10.1074/jbc.ra118.002158

Abstract

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.

Highlights

Conformations adopted by long stretches of single-stranded DNA are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo
Numerous studies have indicated that telomeric G-strand single-stranded DNA (ssDNA) folds into G-quadruplex structures [15, 16, 29]
CD analysis is frequently used to demonstrate the presence of G-quadruplex structures within DNA [30]

Summary

Results

Tandem copies of ssDNA containing the human G-strand (5Ј-TTAGGG-3Ј)n, repeat, the complementary C-strand (5ЈCCCTAA-3Ј)n, repeat, and a mutant repeat disrupting the three-G run (5Ј-TTAGTG-3Ј)n were generated using a ␾29 DNA polymerase rolling circle replication scheme from a minicircle template as previously described (Fig. S1a) [28]. The G-strand ssDNA was subjected to alkaline-agarose gel electrophoresis, and a region corresponding to 1–1.5 knt was isolated, neutralized, and purified This size-selected G-strand DNA (average of 1.25 knt) was prepared for EM in the presence of Kϩ cation, and the length and number of beads were determined for molecules that appeared fully saturated with mostly larger bead-like particles and no significant linker DNA (Fig. 3b, left panel). We first determined that multiple cycles of magnetic extension and release of a single G-strand ssDNA resulted in largely identical force extension curves (Fig. 6a and Fig. S9a) This observation strongly suggests that the G-strand ssDNA spontaneously refolds into comparable stable higher order struc-. We conclude that RAD51 forms a stable nucleoprotein filament in the presence of ATP that fully extends the G-strand ssDNA, unraveling both the embedded G-quadruplex folds and the higher-order G-residue pseudo-fold structures

Discussion

Experimental procedures

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