Dynamics of strand slippage in DNA hairpins formed by CAG repeats: roles of sequence parity and trinucleotide interrupts

Abstract

DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5'-AGCA-3' tetraloops, compared with alternative 5'-CAG-3' triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is "frustrated'' and slips back and forth between states with one TR hanging at the 5' or 3' end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.

Figure 1.

(A) Schematic DNA design for the smFRET analysis of CAG repeat hairpins, with donor in blue and acceptor in red. The CAG sequence in parenthesis is repeated for various hairpins. The hairpin loop of interest is immobilized to a surface by a partial complimentary DNA anchor strand. The TTTTC spacer helps reduce the interaction between the hairpin and junction duplex. (B) CAG hairpins sequences considered in the MD simulations. CG Watson-Crick pairs were added to the ends of short repeats to mimic long canonical stems.

Figure 2.

smFRET analysis and interpretation of A31, (CAG)₁₄ and (CAG)₁₅ hairpin loops, as well as associated hairpin variants. Histograms contain all the timepoints of FRET efficiency from multiple molecules. All the histograms are fit with multiple Gaussian functions (red line) to identify the peak locations. Here we show results for (A) A31; (B) (CAG)₁₄; (C) (CAG)₁₅; (E) (CAG)₁₄T₃; (F) (CAG)₁₅T₃ and (G) (TCC)(CAG)₁₅(GGA). In (D), we present schematic diagrams to show molecular configurations, expected FRET values (upper edge) and the corresponding observed populations (lower edge) of the individual hairpins. The slip designation is indicated below each schematic. (H) Representative smFRET time traces (upper panels, donor signal in blue, acceptor signal in red) and calculated FRET values (lower panels, black) for A₃₁, (CAG)₁₄ and (CAG)₁₅ hairpin loops.

Figure 3.

CAA Interrupts near the loop can stabilize (CAG)₁₅ strand-slipping transitions. (A) Schematics of possible folding configurations and smFRET results for (CAG)₁₅ and several CAA interrupted (CAG)₁₅ TR hairpins. Sequences are indicated at the top of the diagram. Slip designation and observed populations are indicated along the lower edge. Results of (CAG)₁₅ experiment from Figure 2 are listed for comparison. (B–D) Histograms of smFRET measurements for the hairpins diagramed in (A). (E) Representative time traces (upper panels, donor signal in blue, acceptor signal in red) and calculated FRET values (lower panels, black) for the (CAG)₁₅ and the CAA interrupted (CAG)₁₅ TR hairpins.

Figure 4.

(A) The sheared C·G pair with a C(O2)–G(H1) H-bond in the stable clamped triloop of CAG1 (Figure 1). (B) A side view of the CAG1 triloop with C3(blue), A4(orange) and G5(cyan). (C and D) Two long-lived loop conformations for CAG4 (Figure 1): (C) S(aa)-L(as)-G(a) (stable) and (D) S(as)-L(ss)-G(s) (kinetic trap). The cartoon only shows the residues from A3 to A12 with A6(blue), G7(orange), C8(red), A9(cyan) and A3–A12(green). (E) The strong stacking between the A–A mismatch in the tetraloop of CAG4 and the C–G Watson–Crick basepair immediately below: C5(yellow)/A6(blue) and A9(cyan)/G10(pink) in the S(aa)-L(as)-G(a) conformation (bases in the 5′-AGCA-3′ tetraloop in conformation anti–anti–anti–syn, with anti–anti conformation for the first mismatch closer to the loop, see text). (F) The two-stack loop in L(aa) (A–A in 5′-AGCA-3′ in anti-anti) for CAG2. Bases in colors: A3(blue), G4(orange), C5(red) and A6(cyan). (G) View for (F) of the stacking of A3(blue)/G4(orange) and C5(red)/A6(cyan).

Figure 5.

Results for the cluster-like analysis of different conformations for the AGCA tetraloop in the CAG4 hairpin. Different conformations are represented as different colors. Three different kinds of two-stack loops are shown in different shadows. The areas of the circles are representative of the percentage of time that the system spends in that conformation. Likewise the arrows indicate the observed transitions during the simulation with the width of the arrow representing the estimated frequency of transitions. Favorable conformations are indicated by large, growing circles. The simulation results show that the L(as)–G(a) (red; AGCA in anti–anti–anti–syn conformation) and the two versions of L(aa)–G(a) (blue, gray with horizontal line shading; AGCA all in anti conformation) are the most favorable conformations, as discussed in the text.