Oligodeoxynucleotide binding to (CTG) · (CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability

Abstract

(CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.

Fig 1

ODNs release replication fork stalling. (a) Diagram of ectopic c-myc–TNR locus in (CTG)₄₅ and (CAG)₄₅ cell lines. Horizontal line, chromosomal DNA; oval, the bidirectional c-myc replicator; dashed arrows, bidirectional replication forks; vertical rectangle, (CTG)₄₅ repeat (above chromosomal DNA) or (CAG)₄₅ repeat tract (below chromosome) [in the (CTG)₄₅ cell line, (CTG)₄₅ is in the lagging-strand template; in the (CAG)₄₅ cell line, (CAG)₄₅ is on the lagging-strand template]; short horizontal bars above the chromosomal DNA, locations of STS-pVD1 and STS-pVD2. P1 and P2 primers for small-pool PCR are indicated by two head-on arrows. (b) HeLa derivative cell lines HeLa/c-myc, HeLa/c-myc–(CTG)₄₅, and HeLa/c-myc–(CAG)₄₅ were transfected with the indicated ODNs every 48 h for three times, and nascent DNA was isolated as described previously (31, 57). Size-fractioned nascent DNA was isolated and quantitated using Sybr green real-time PCR (Life Technologies). For different nascent DNA preparations, STS-54.8 at the β-globin origin (31) was used as the internal normalizer. To compare the nascent DNA abundance at STS-pVD1 and STS-pVD2, data from WT were set equal to 100%, and the nascent DNA abundances of (CTG)₄₅ and (CAG)₄₅ cells with different treatments at the same STS were accordingly calculated. The data presented are the means (±standard deviations) of triplicate analyses on at least three independent preparations of nascent DNA from each cell line with proper treatments. Error bars, standard deviations.

Fig 2

ODNs inhibit hairpin formation on lagging-strand templates. Cells were cotransfected with ZFN (or ZFP) expression plasmids and either a random 21-mer ODN (mock-treated ODN) or ODNs every 48 h for three times before genomic DNA was isolated. spPCR products of genomic DNA were amplified by primers P1 and P2 (Fig. 1a) and resolved by 8% PAGE. A 100-bp DNA ladder was used as the DNA size marker. (a) Inhibition of hairpin formation by (CAG)₇ in (CTG)₄₅ cells. Lanes 1 to 4, mock ODN treatment of cells expressing ZFP_CTG; lanes 5 to 8, mock ODN treatment of cells expressing ZFN_CTG; lanes 9 to 12, (CTG)₇ treatment of cells expressing ZFP_CTG; lanes 13 to 16, (CAG)₇ treatment of cells expressing ZFN_CTG. The ∼270-bp PCR product [(CTG)₄₅ · (CAG)₄₅ progenitor allele] is indicated with an arrow. The lower-mobility shadow bands observed above the ∼270-bp amplification product of the (CTG)₄₅ · (CAG)₄₅ progenitor sequence are slipped-strand structures formed in vitro during PCR reannealing (6), as demonstrated by reamplification of the linear ∼270-bp product band (8). Bands migrating faster than the progenitor product are the results of in vivo nuclease cleavage. (b) Predicted hairpin inhibition by the (CAG)₇ ODNs. (c) Inhibition of hairpin formation by (CTG)₇ in (CAG)₄₅ cells. Lanes 1 to 4, mock ODN treatment of cells expressing ZFP_CAG; lanes 5 to 8, mock ODN treatment of cells expressing ZFN_CAG; lanes 9 to 12, (CTG)₇ treatment of cells expressing ZFP_CAG; lanes 13 to 16, (CTG)₇ treatment of cells expressing ZFN_CAG. (d) Predicted hairpin inhibition by (CTG)₇ in (CAG)₄₅ cells. In panels b and d, (CTG)_n, ZFN_CTG, (CAG)_n, and ZFN_CAG are depicted in gray. ODNs are depicted as multiple short bars, and ZFNs are depicted as sickles.

Fig 3

ODNs do not inhibit hairpin formation on the leading-strand template. (a) Inability of (CTG)₇ to inhibit hairpin formation in (CTG)₄₅ cells. Lanes 1 to 4, mock ODN treatment of cells expressing ZFP_CAG; lanes 5 to 8, mock ODN treatment of cells expressing ZFN_CAG; lanes 9 to 13, (CTG)₇ treatment of cells expressing ZFP_CAG; lanes 14 to 18, (CTG)₇ treatment of cells expressing ZFN_CAG. spPCR products of genomic DNA were amplified by primers P1 and P2 (Fig. 1a) and resolved by PAGE. (b) Predicted hairpin inhibition by (CTG)₇. (c) Absence of inhibition of hairpin formation by (CAG)₇ in (CAG)₄₅ cells. Lanes 1 to 4, control ODN treatment of cells expressing ZFP_CTG; lanes 5 to 8, control ODN treatment of cells expressing ZFN_CTG; lanes 9 to 12, (CAG)₇ treatment of cells expressing ZFP_CTG; lanes 13 to 16, (CAG)₇ treatment of cells expressing ZFN_CTG. (d) Predicted hairpin inhibition by (CAG)₇ in (CAG)₄₅ cells.

Fig 4

ODN-mediated inhibition of lagging-strand template hairpins suppresses hairpin formation on leading-strand templates. (a) Simultaneous inhibition of hairpin formation on both lagging- and leading-strand templates by (CAG)₇ in (CTG)₄₅ cells. Lanes 1 to 4, (CAG)₇ treatment of cells expressing ZFP_CAG; lanes 5 to 8, (CAG)₇ treatment of cells expressing ZFN_CAG; lanes 9 to 12, (CTG)₇ treatment of cells expressing ZFP_CTG; lanes 13 to 16, (CTG)₇ treatment of cells expressing ZFN_CTG. spPCR products of genomic DNA were amplified by primers P1 and P2 (Fig. 1a) and resolved by PAGE. (b) Predicted hairpin inhibition by (CAG)₇ and (CTG)₇. (c) Simultaneous inhibition of hairpin formation on both lagging- and leading-strand templates by (CTG)₇ in (CAG)₄₅ cells. Lanes 1 to 4, (CTG)₇ treatment of cells expressing ZFP_CTG; lanes 5 to 8, (CTG)₇ treatment of cells expressing ZFN_CTG; lanes 9 to 12, (CAG)₇ treatment of cells expressing ZFP_CAG; lanes 13 to 16, (CAG)₇ treatment of cells expressing ZFN_CAG. (d) Predicted hairpin inhibition by (CTG)₇ in (CAG)₄₅ cells.

Fig 5

ODNs stabilize (CTG)₄₅ · (CAG)₄₅ TNRs against emetine-induced instability. (CTG)₄₅ or (CAG)₄₅ cells were treated with emetine (EME) (a) or aphidicolin (APH) (b) and transfected with the indicated ODNs or not transfected (see Materials and Methods). spPCR products of genomic DNA were amplified by primers P1 and P2 (Fig. 1a) and resolved by PAGE. Arrows, progenitor PCR products; exp., expansion; contr., contraction; lanes M, molecular size markers.

Fig 6

ODNs inhibit (CTG)₄₅ · (CAG)₄₅ TNR instability during unperturbed cell culture. (CTG)₄₅ · (CAG)₄₅ cells were grown for ∼25 or ∼250 PDs in the absence or presence of the indicated lagging-strand-targeting ODNs. spPCR products of genomic DNA were amplified by primers P1 and P2 (Fig. 1a) and resolved by PAGE. Arrows, progenitor PCR products; exp., expansion; contr., contraction; lanes M, molecular size markers.

Fig 7

Model of coordinated hairpin formation. (a) Lagging-strand hairpin formation during unperturbed cell division causes polymerase (circle) stalling or fork reversal. (b) ODNs prevent lagging-strand hairpin formation by annealing to the lagging-strand template (23, 25) or reversing hairpin formation by annealing to the hairpin (21, 33). (c) Slowing of leading- and lagging-strand polymerases leads to fork reversal which cannot be reversed by ODNs binding to the lagging-strand template. (d) In the absence of ODNs, fork reversal leads to hairpin formation on leading- and lagging-strand templates (generating contractions) and leading- and lagging-strand nascent DNA (generating expansions). Gray lines, TNR sequences; black lines, flanking DNA. For brevity, template and nascent-strand hairpins are shown at the same fork (c and d), although these structures need not occur in the same cell.