On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability

Abstract

Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?

Keywords: DNA recombination; DNA repair; DNA replication; DNA structure; G-quadruplex; Huntington disease; R-loop; S-DNA; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); gene expression; genomic instability; hairpin; trinucleotide repeat disease; triplex H-DNA.

Figure 1.

Loss-of-function mechanisms caused by expanded repeats exemplified by GAA repeats in FXN and CGG repeats in FMR1 genes. Repeats are shown in blue and red, and the flanking DNA sequences are shown in black. Top, when repeats are short, transcription is unimpeded. Bottom left, expansion of GAA repeats leads to formation of an R-loop at the repeat, which triggers histone deacetylation and the presence of repressive histone marks (such as histone H3 Lys-9 methylation), heterochromatization, and gene silencing (reviewed in Ref. 76). Bottom right, expansion of CGG repeats leads to formation of an R-loop at the repeat, which triggers methylation of the repeat as well as of the CpG islands upstream of the FMR1 promoter, followed by histone methylation. This results in heterochromatization and, ultimately, gene silencing (reviewed in Ref. 81).

Figure 2.

Toxic gain-of-function mechanisms in REDs exemplified by an expansion of a CTG repeat. Left, transcription of expanded CTG repeats produces long r(CUG)_n RNA species that fold into RNA secondary structures, aggregate, and sequester the Muscleblind protein. This is the main source of toxicity in DM1 disease (reviewed in Ref. 15). Right, RAN translation of r(CUG)_n. Expanded RNA repeats recruit ribosomes. This recruitment is likely mediated by the formation of an RNA secondary structure. Translation of r(CUG)_n results in accumulation of toxic repetitive polypeptides in all three reading frames (reviewed in Refs. 123–125). Note that antisense transcription of the same repeat might also undergo RAN translation (not shown).

Figure 3.

Types of dynamic DNA structures and repeat sequences that form them. *, complement of CCTG; **, complement of CCCTCT.

Figure 4.

A, DNA cruciform versus S-DNA structures. B, difference in the kinetics of perfect versus imperfect hairpin formation (see “Imperfect hairpins” in the “Dynamic DNA structures as the key to repeat instability” section).

Figure 5.

Two models depicting the potential role of Rad27 flap endonuclease in the instability of structure-forming repeats. A, flap ligation model. In the absence of Rad27, long flaps that formed during Okazaki maturation might become incorporated into the nascent DNA during lagging strand synthesis, resulting in repeat expansion. B, RPA depletion model. In the absence of Rad27, long flaps that accumulate genome-wide during Okazaki fragment maturation titrate RPA out from the repetitive DNA. This promotes formation of DNA secondary structures (the hairpin is shown) within the repeat and can result in repeat contraction or expansion.

Figure 6.

Possible mutagenic consequences of fork stalling within structure-forming repeats (see “Direct evidence that expandable repeats are hard to replicate” for details).

Figure 7.

Various R-loop-based structures formed by expandable DNA repeats (see “Role of transcription in repeat instability” for details).

Figure 8.

HR mechanisms implemented in repeat instability. A, as a result of an unequal crossing over during meiosis, one homologous chromosome inherits a contracted repeat tract, whereas another inherits an expanded repeat tract. This mechanism is typical for poly(A) diseases. B, a DSB initiated at a repetitive sequence is followed by end resection and reannealing of the repetitive ends. This can ultimately result in repeat contraction. C, a one-ended DSB can be repaired via BIR. Out-of-register invasion into a repetitive template can give rise to both expansions and contractions, while point mutations accumulated in the course of BIR are responsible for the RIM phenomenon.

Figure 9.

Two hypothetical hybrid DNA repair pathways that could promote repeat instability. A, an oxidized nucleotide within a repetitive tract is being removed by the BER pathway to create a nick. In the course of strand displacement synthesis, the displaced flap might form a DNA secondary structure (e.g. a hairpin). The structure is stabilized by MutSβ and gets incorporated into the DNA, resulting in a repeat expansion. B, when a repeat is located in an actively transcribed gene, RNA polymerase might promote formation of a DNA secondary structure, such as S-DNA. These structures, when additionally stabilized by MutSβ, stall the next RNA polymerase, triggering NER. The NER repair might lead to either excision or incorporation of a secondary structure, leading to repeat contraction (this scenario is shown) or expansion (not shown), respectively.