Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

Abstract

Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington's Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets.

Fig. 1. Two-state FRET dynamics for static 3WJs.

a Static 3WJs, S1–S4, formed from two DNA strands, labeled with either Alexa488 (green) or Cy5 (red); the (CTG)₁₀ slip-out (blue) is fixed relative to the fully complementary dsDNA (black). b FRET histograms from MFD of freely diffusing 3WJs fitted to one donor-only state and one (S4) or two (S1–S3) FRET states. c TIRF time traces of surface-immobilized 3WJs; Alexa488 (green) and Cy5 (red) intensities, and calculated FRET efficiency (E_FRET) and idealized trace (red) from Hidden Markov modeling. d Transition density plots of TIRF FRET traces. Measured at 20 °C in a buffer containing 1 mM MgCl₂.

Fig. 2. Interconversion of positional isomers for mobile 3WJs.

a The presence of three complementary CAG (orange) and CTG (blue) repeats in the duplex and 10 CAG repeats in the slip-out (orange) could result in four positional isomers P1–P4. b 2D MFD plot shows FRET efficiency (E_FRET) or donor anisotropy (r_D) vs donor lifetime [τ_D(A)] for freely diffusing 3WJs. c MFD FRET histogram fits to six FRET states. d TIRF time traces of surface-immobilized 3WJs; Alexa488 (green) and Cy5 (red) intensities, and calculated FRET efficiency (E_FRET). e TIRF FRET histogram fitted to six FRET states. Measured at 20 °C in a buffer containing 1 mM MgCl₂.

Fig. 3. Dependence of dynamics on repeat length.

Transition density plots from TIRF time traces of 3WJs with three complementary CAG (orange) and CTG (blue) repeats in the duplex and (CAG)_n slip-outs (n = 4, 6, 8, 10, 20, and 30). 3WJs with slip-outs of (CAG)₄ have two FRET states, while all other 3WJs are fitted to six FRET states. Similar plots are found for 3WJs with (CTG)_n slip-outs (Supplementary Fig. 20). Measured at 20 °C in a buffer containing 1 mM MgCl₂.

Fig. 4. Conformational and branch migration dynamics in slip-out 3WJs.

a 3WJs with hairpin slip-outs can undergo conformational change, attributed to branchpoint rearrangement (e.g., expansion/contraction). b Kinetic scheme showing the eight possible FRET states as a result of the two conformations for each positional isomer. c Selected rate constants for mobile 3WJs with 10, 20, or 30 repeats in the slip-out. Error bars represent the standard error from non-linear curve fitting (see Methods for details). d Cumulative dwell times histogram for static 3WJs (left), and mobile 3WJs with CAG (middle) or CTG (right) slip-outs. e Model of branch migration in 3WJs with trinucleotide repeats in a slip-out and in the duplex. Measurements made at 20 °C in a buffer containing 1 mM MgCl₂.