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. 2012 Feb 1;11(2):201-9.
doi: 10.1016/j.dnarep.2011.10.020. Epub 2011 Oct 29.

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Tianyi Zhang  1 , Jian Huang, Liya Gu, Guo-Min Li
Affiliations

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Tianyi Zhang et al. DNA Repair (Amst). .

Abstract

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.

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Conflict of interest statement

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Construction of DNA heteroduplex substrates containing different sizes of CAG/CTG hairpins. (A) Oligonucleotides containing different numbers of CAG/CTG repeats were cloned into the HindIII and EcoRI restriction enzyme sites of an M13mp18 phage derivative. Heteroduplex substrates were constructed as described [13]. All substrates contain a single strand nick in the complementary strand generated by BglI digestion prior to heteroduplex formation. (B) Substrates with different sizes of CAG or CTG hairpins were constructed by heteroduplexing DNA containing different numbers of CAG and CTG repeats in the complementary and viral strands. Predicted structures are shown to the right of the sequences. Repeat sequences are indicated as red (CTG) and blue (CAG).
Fig. 2
Fig. 2
Repair of (CAG)n and (CTG)n substrates in HeLa cell extracts. (A–D, top) The indicated (CAG)n and (CTG)n hairpin substrates (200 ng) were incubated with (+) or without (−) 100 μg of HeLa nuclear extracts under the normal HPR conditions (see Section 2. Repaired products were analyzed by Southern blotting using a probe close to the BglI site of the nicked strand (red bar on the substrate structure). Repair products are highlighted by red boxes. (A–D, bottom) Repair efficiencies of the different (CAG)n and (CTG)n substrates. Repair efficiencies are quantified from at least three individual assays and error bars represent the standard error of the mean.
Fig. 3
Fig. 3
Influence of MutSα and MutSβ on smaller hairpin repair. HPR assays were performed as described in Fig. 2 legend, but in the presence or absence of purified MutSα or MutSβ. (A) V-(CTG)n substrates. The upper bands are repair products, and lower bands are substrates. (B) C-(CAG)n substrates. The lower bands are repair products, while the upper bands are substrates.
Fig. 4
Fig. 4
Time course analysis of repair intermediates of C-(CAG)n substrates. The incision/excision time course assays were performed under the HPR conditions, but in the absence of dNTPs. DNA samples were recovered and subjected to Southern blot analysis using the PstI probe, which detects both incisions and excisions. (A) Substrate C-(CAG)15. (B) Substrate C-(CAG)10. (C) Substrate C-(CAG)5. Red rectangles indicate excision intermediates. Black arrows indicate the products generated by a flap endonuclease.
Fig. 5
Fig. 5
Analysis of repair intermediates of C-(CTG)n substrates. (A) C-(CTG)5 incision/excision time course assay probed with the BglI probe. (B) C-(CTG)5 incision/excision time course assay probed with the PstI probe. (C) Determination of incision sites for substrate C-(CTG)25. The left panel shows the size markers generated from (CTG)35 viral DNA by DNA sequencing analysis (only the T reaction shown). The right panel shows the incision intermediates derived from substrate C-(CTG)25 after incubation with HeLa nuclear extracts for 30 min. (D) Determination of incision sites for substrate C-(CTG)5. The left panel shows the size markers generated from (CTG)15 viral DNA by DNA sequencing analysis (only the T reaction shown). The right panel shows the incision intermediates derived from substrate C-(CTG)5 after incubation with HeLa nuclear extracts for 5 min. (E) C-(CTG)15 incision/excision time course assay probed with PstI prode. (F) C-(CTG)10 incision/excision time course assay probed with PstI probe. Red rectangles indicate excision products; red arrows indicate endonuclease products; and blue arrows represent flap endonuclease products.
Fig. 6
Fig. 6
Analysis of repair intermediates of V-(CAG)n and V-(CTG)n substrates. (A–C) Incision/excision time course assays for all V-(CAG)n substrates. (D–F) Incision/excision time course assays for all V-(CTG)n substrates. All assays were probed with the BglI probe. Red arrows indicate endonuclease products. Red rectangles indicate further degradation of the incision products.
Fig. 7
Fig. 7
Incision locations for V-(CAG)n and V-(CTG)n substrates. (A) Size markers were generated from (CAG)15 and (CAG)10 viral DNAs. V-(CAG)n substrates were incubated with HeLa extracts for 15 min. (B) Sequence markers were generated from (CTG)15 and (CTG)10 viral DNAs. V-(CTG)n substrates were incubated with HeLa extracts for 15 min. The assays were probed with the BglI probe.
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