Interconverting conformations of slipped-DNA junctions formed by trinucleotide repeats affect repair outcome

Abstract

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations.

Figure 1

Slipped DNA junctions formed by (CTG)·(CAG) repeats. (A and B) Slipped DNAs are composed of three arms, two made of complementary repeat strands and the third being the CAG (blue) or CTG (red) repeat slip-out. Slip-outs assume intrastrand hairpins, and their structural characterizations have been extensive.⁻ Possible junction conformations include fully paired strands at the slip-out or those with one or two unpaired bases at the junction, where the latter can potentially interconvert with the fully paired form. The angles displayed between the junction arms are not meant to reflect the actual angles observed in the three-way junctions. In panel A, unpaired nucleotides are highlighted in green; in panel B, nucleotides with the potential to be unpaired are shown in green boxes. (C) Electrophoretic migration of slipped-junction species was slower than expected. DNAs are a single species on denaturing gels (Figure S3 of the Supporting Information).

Figure 2

NMR data reveal that slipped junctions form multiple interconverting conformations. (A–C) NMR spectra for J4/5. (A) T Me–T H6 section of the 2D TOCSY spectrum in D₂O at 20 °C. (B) Imino to T Me and (C) imino to imino sections of the 600 MHz 2D NOESY spectrum of J4/5 recorded in H₂O at 10 °C with a mixing time of 300 ms. For an explanation of the J4/5 NMR spectra in panels A–C, see the text and a detailed description in section V of the Supporting Information. In panel A, the cross-peaks at characteristic chemical shift positions of the T’s in the two CTTG tetraloops that cap two arms (panel D) are labeled with their residue numbers (T₁₃, T₁₄, T₂₆, and T₂₇). The other cross-peak resonances in panel A are numbered from 1 to 15; cross-peak 15 can be assigned to T₃₈. In panels B and C, the imino resonances from T’s in A:T base pairs are numbered from 1 to 4. Except for number 1, which shows no cross-peaks to G imino resonances (panel C), all these imino resonances show two cross-peaks to G iminos and are thus from A:T base pairs flanked by two C:G base pairs. The presence of multiple conformations is indicated by, for instance, the exchange cross-peaks in panel C (see, e.g., the encircled cross-peak between 2 and 3 labeled exchange in the bottom left of panel C near the diagonal; note that in NOESY spectra, the intensities of symmetry-related cross-peaks are not always equal), and the multiple imino–methyl contacts in panel B (see the text). Imino resonance 4 can be assigned to T₃₈ because of the contact with the T₃₈ Me seen in panel B (see the text). Imino resonance 3 includes more than two imino protons and/or multiple conformations as described in the text. (D–F) Schematics of conformations of J4/5, J1/2, and J3, respectively, that are consistent with their NMR spectra (NMR detailed in Figures S5–S7, Tables S3, and sections V–VII of the Supporting Information). In panel D, species J5, J4/5_1, and J4/5_2 are the J5 variants outlined in Figure 1A. In panel E, species J2, J1/2_1, and J1/2_2 are the J2 variants outlined in Figure 1A. In panel F, species J3, J3_1, and J3_2 are the J3 variants outlined in Figure 1A. The CAG repeats are colored blue and the CTG repeats red. The locations of pertinent nucleotides are indicated by their sequence order. The most likely stacking of the arms in the three-way junctions is shown using published arm nomenclature I, II, and III (Figure S4 of the Supporting Information). Stacking preferences are based on published loop and pyrimidine rules.^,

Figure 3

Electrophoretic mobility shift assays with [γ-³²P]ATP-end-labeled junction substrates J1/2, J3, J4/5, and J6. (A) Binding of purified human HMGB1 (0, 0.1, 1, and 10 pmol) to 100 fmol of each [γ-³²P]ATP-end-labeled junction substrate. (B) Binding of purified bacterial MutS (0, 0.1, 1, and 10 pmol) to 100 fmol of each [γ-³²P]ATP-end-labeled junction substrate. (C) Binding of purified human MutSα and MutSβ [0 (−), 250 (+), and 750 fmol (++)] to 50 fmol of each [γ-³²P]ATP-end-labeled junction substrate. (D) Cleavage of [γ-³²P]ATP end-labeled junction substrates J1/2, J3, J4/5, and J6 by human XPG and ERCC1-XPF nucleases. XPG and ERCC1-XPF proteins (100 fmol) were incubated with 100 fmol of each [γ-³²P]ATP-end-labeled junction substrate as indicated. EMSA and cleavage products were run on a 6% polyacrylamide gel that was dried and exposed to X-ray film. Arrows indicate the cleavage product for each junction substrate. Cleavage site mapping is shown in Figure S8 of the Supporting Information.

Figure 4

Repair of junction structures. (A) Junction sequences of the circular DNA repair substrates. Slip-outs are defined by both the nucleotides in the slip-out stem and the (CNG)_n noted above the slip-out. Where applicable, only one possible unpaired nucleotide form is shown; other nucleotides with the potential to be unpaired are shown in green boxes. (B) DNA repair substrates modeling possible junction structures. Circular heteroduplexes with slipped (CNG)₁₇·(CNG)_0/1/2 repeats modeling intermediates of expansions with nicks in the slipped CAG or CTG strand. Substrates are named on the basis of the junctions (see Figure 1) that they model: p-J1 is a circular substrate modeling junction J1. (C) Southern blot analysis of repair of circular substrates using HeLa extracts. “Starting material” indicates unprocessed heteroduplexes, with the background band. The background band seen at varying levels in the starting material is the remaining double-stranded DNA from the heteroduplexing reaction mixture and cannot be completely eliminated; however, the amount of background is irrelevant as the starting material was subtracted from the repair efficiencies for quantification (as previously described^,). (D) Circular substrate repair efficiencies (corrected for starting material). Nick-directed repair is shown as hatched bars, and non-nick-directed repair is shown as solid bars; error bars represent the standard deviation. Efficiency values are based on at least three replicates. The dashed red line indicates typical levels of non-nick-directed repair, as seen for a G-T mismatch. Nick-directed repair: p-J1 vs p-J3, p < 0.01; p-J1 vs p-J1/2, p < 0.01; p-J3 vs p-J1/2, p < 0.05; p-J4/5 vs p-J4, p < 0.05; p-J4/5 vs p-J6, p < 0.05. Non-nick-directed repair: p-J1 vs p-J3, p < 0.05; p-J3 vs p-J1/2, p < 0.05; p-J6 vs p-J4/5, p < 0.05. p values determined by t test (n ≥ 3).

Figure 5

Nick-directed vs non-nick-directed repair. Correct repair is always nick-directed, involving excision of the excess repeats and gap filling using the continuous strand as a template; non-nick-directed repair leads to retention of the excess repeats by using the nicked slipped strand as the template for gap filling, leading to an expansion mutation product.