Isolated short CTG/CAG DNA slip-outs are repaired efficiently by hMutSbeta, but clustered slip-outs are poorly repaired

Abstract

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntington's disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSbeta. Higher or lower levels of hMutSbeta or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50*(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSbeta requirement for instability, hMutSbeta is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each other's repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSbeta. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions.

Fig. 1.

Repair depends on slip-out size and MMR. Circular hybrids with slipped (CTG)x•(CAG)y repeats (x and y are 30, 47, 48, or 50 repeats, and Δ = x – y = 20, 3, or 1 repeats) modeling intermediates of expansions with nicks in slipped-strand (SI Appendix, Fig. S1). (A) MMR-proficient (HeLa) and MMR-deficient (LoVo) extracts (90 μg) in 50-μL reactions, 22 fmol of circular substrate, were incubated for 30 min. Repair efficiencies were calculated for three to six replicates (Materials and Methods). G-T mismatch is an MMR-dependent control. Graph shows starting background (chequered bars) and repair (white bars). (B) (Upper) Southern blot analysis of repair of a single CTG slip-out, processed by 90-μg LoVo extracts with and without 250 ng recombinant MutSβ or MutSα. (Lower) Repair efficiencies were calculated from three to six replicates. Graph shows starting background (chequered bars) and repair (white bars).

Fig. 2.

Short slip-out repair is sensitive to MutSβ concentration. (A) hMSH3, hMSH6, and hMSH2 proteins in cell extracts. In lanes 1–4, 50 μg of HeLa, HeLa^MTXR1, HeLa^MTXR2, or LoVo cell extracts, respectively, were separated by SDS/PAGE. In lanes 5–10, 15, 120, or 240 ng of purified hMutSα or 7, 30, or 455 ng of purified hMutSβ were loaded to demonstrate the similar immune responses of the proteins and the lower detection limits of the experiment. Western blotting was simultaneous for hMSH3, hMSH6, hMSH2, and actin (SI Appendix, Fig. S3). DHFR was blotted and probed separately. (B) (Upper) Southern blot analysis of repair of a circular hybrid with a single CTG slip-out, processed by HeLa, HeLa^MTXR1, and HeLa^MTXR2 with the starting material shown on the left. Autorad is from a single gel, identical exposure time, with intervening lanes excised for presentation. (Lower) Repair efficiencies of corresponding reactions were calculated on three to six replicates. Graph shows starting background (hatched bars) and repair (white bars). (C) (Upper) Southern blot analysis of repair of a circular hybrid with a single CTG slip-out, processed with mixtures of hMSH3-overexpressed HeLa^MTXR1, and MMR-deficient LoVo extracts to dilute hMSH3 levels. (Lower) Repair efficiencies were calculated on three to six replicates. Graph shows starting background (hatched bars) and repair (white bars).

Fig. 3.

Multiple slip-outs interfere with repair. Circular hybrids with homoduplex slipped (CTG)50•(CAG)50 S-DNAs with a unique nick, copurified with fully duplexed forms (background) (SI Appendix, Fig. S1). (Upper) Southern blot analysis of S-DNA repair products using MMR-proficient HeLa and MMR-deficient LoVo extracts. Autorad is from a single gel, identical exposure time, with intervening lanes excised for presentation. (Lower) Repair efficiencies were calculated from three to six replicates. Graph shows starting background (hatched bars) and repair (white bars). See also Figs. S6 and S9 in SI Appendix.

Fig. 4.

MMR and slip-out structure-dependent repair outcomes. (A) Summary of reparability data and MMR dependence of SI-DNA and S-DNA conformations. See also Fig. S9 in SI Appendix. (B) Proposed model (see text).