Trinucleotide repeat DNA alters structure to minimize the thermodynamic impact of 8-oxo-7,8-dihydroguanine

Abstract

In the phenomenon of trinucleotide repeat (TNR) expansion, an important interplay exists between DNA damage repair of 8-oxo-7,8-dihydroguanine (8-oxoG) and noncanonical structure formation. We show that TNR DNA adapts its structure to accommodate 8-oxoG. Using chemical probe analysis, we find that CAG repeats composing the stem-loop arm of a three-way junction alter the population of structures in response to 8-oxoG by positioning the lesion at or near the loop. Furthermore, we find that oligonucleotides composed of odd-numbered repeat sequences, which form populations of two structures, will also alter their structure to place 8-oxoG in the loop. However, sequences with an even number of repeats do not display this behavior. Analysis by differential scanning calorimetry indicates that when the lesion is located within the loop, there are no significant changes to the thermodynamic parameters as compared to the DNA lacking 8-oxoG. This contrasts with the enthalpic destabilization observed when 8-oxoG is base-paired to C and indicates that positioning 8-oxoG in the loop avoids the thermodynamic penalty associated with 8-oxoG base-pairing. Since formation of stem-loop hairpins is proposed to facilitate TNR expansion, these results highlight the importance of defining the structural consequences of DNA damage.

Figure 1

Autoradiogram of DEPC reactions with (A) CAG16-TWJ/CTG6-TWJ and (B) CAG16-TWJ-8OX/CTG6-TWJ. Lanes 1 are DNA alone, lanes 2 are piperidine-treated DNA, and lanes 3-5 are DNA treated with 2.5% (v/v) DEPC at 37 °C for 5, 15 or 30 min respectively, followed by treatment with piperidine. Lanes A are a marker created by treatment of the single-stranded CAG16-TWJ or CAG16-TWJ-8OX substrate with 1% DEPC at 37 °C for 15 min. The proposed structures derived from the reactivity of each substrate are shown to the right of the autoradiogram.

Figure 2

(A) Autoradiogram of DEPC reactions with CAG10, CAG10-5OX, and CAG10-6OX. Lanes 1 are DNA alone, lanes 2 are piperidine-treated DNA, and lanes 3-5 are DNA treated with 1% (v/v) DEPC at 37 °C for 5, 15 or 30 min respectively, followed by treatment with piperidine. Histograms of DEPC reactivity, derived from lanes 4, with (B) CAG10, (C) CAG10-5OX, and (D) CAG10-6OX. The dashed line in each histogram demarks the level of reactivity of adenines in the stem region, which serves as a baseline to establish hyper-reactivity of the nucleobases in the loop. Proposed structures for CAG10, CAG10-5OX, and CAG10-6OX are shown next to the corresponding histogram. The closed wedge indicates hyper-reactivity to DEPC and the open wedge indicates moderate hyper-reactivity to DEPC.

Figure 3

Histograms displaying reactivity of CAG7, CAG11, and CAG15 to (A) DEPC and (B) DMS. Histograms are derived from lanes 4 of the autoradiograms shown in Supporting Information Figure S3, in which the DNA was treated with the chemical probe at 37 °C for 15 min. The dashed line demarks the level of reactivity of adenines or guanines in the stem region, which serves as a baseline to establish hyper-reactivity of nucleobases in the loop. The closed wedge indicates the greatest degree of hyper-reactivity to DEPC, the open wedge indicates moderate hyper-reactivity to DEPC, and the arrow indicates hyper-reactivity to DMS. (C) Two potential scenarios for the structures of CAG7, CAG11, and CAG15 derived from their reactivity to DEPC and DMS. Structures containing a seven nucleobase loop and blunt-ended stem are shown in C(i) while a population of two stem-loops each containing a four nucleobase loop and either a 5′ or 3′ overhang is shown in C(ii).

Figure 4

(A) Proposed structures for CAG7-3′Overhang (left) and CAG7-5′Overhang (right) and (B) CAG7-Blunt Stem. Histograms displaying reactivity to DMS for (C) CAG7-3′+5′Overhangs, (D) CAG7-Blunt Stem, and (E) and CAG7. Histograms are derived from lane 4 of the autoradiograms shown in Figure S4 and S5 where the DNA was treated with 0.05% (v/v) DMS at 37 °C for 15 minutes. The arrow indicates hyper-reactivity to DMS.

Figure 5

(A) Autoradiogram of DEPC reactions with CAG11, CAG11-5OX, and CAG11-6OX. Lanes 1 are DNA alone, lanes 2 are piperidine-treated DNA, and lanes 3-5 are DNA treated with 1% (v/v) DEPC at 37 °C for 5, 15 or 30 min respectively, followed by treatment with piperidine. Histograms of DEPC reactivity, derived from lanes 4, with (B) CAG11, (C) CAG11-5OX, and (D) CAG11-6OX. The dashed line in each histogram demarks the level of reactivity of adenines in the stem region, which serves as a baseline to establish hyper-reactivity of the nucleobases in the loop. Proposed structures for CAG11, CAG11-5OX, and CAG11-6OX are shown next to the corresponding histogram. The closed wedge indicates the greatest degree of hyper-reactivity to DEPC and the open wedge indicates moderate hyper-reactivity to DEPC.

Figure 6

Representative thermograms, both background corrected using a buffer-buffer scan and baseline corrected using a polynomial fit, for (A) CAG7 at 33.1 μM, (B) CAG10 at 30 μM, (C) CAG11 at 16.8 μM, and (D) CAG15 at 40 μM. All samples were suspended in buffer containing 20 mM sodium phosphate, 70 mM NaCl, pH 7.5.