Repeat instability during DNA repair: Insights from model systems

Abstract

The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them.

Keywords: Chromosome fragility; DNA damage checkpoint; DNA structure; recombination; repair; replication; structure-specific helicases; trinucleotide repeat expansion.

Figure 1

Stable non-B form DNA structures formed by expandable repeats. (A) Hairpins can be formed by G–C-rich expanding triplet repeats with stability: CGG>CCG>CTG>CAG. (B) A slipped-strand structure occurs when hairpins form simultaneously on both strands. (C) RNA/DNA hybrids (R-loops) occur during transcription when the newly synthesized RNA transcript stably pairs with single-stranded DNA in the transcription bubble. Purine-rich repeats are especially prone to forming persistent R-loops. (D) G-quadruplex structures can be formed by CGG repeats and G₄C₂ repeats. G-quadruplexes can be parallel (not shown) or anti-parallel (shown). (E) Triple helical DNA structures form at GAA/TCC repeats and can be either the purine:purine:pyrimidine triplexes or pyrimidine:purine pyrimidine triplexes. (F) AT-rich ATTCT/AGAAT repeats are DNA-unwinding elements, melting the double helix to form a region of unpaired DNA.

Figure 2

General mechanisms of trinucleotide repeat instability. (A) Polymerase slippage can lead to formation of secondary structures on the extending nascent strand (expansion) or template strand (contraction). Slippage can also occur at replication barriers, and structures can form on the nascent stands or template strands at the stalled replication fork. Reversed forks can be processed into a hairpin on the leading strand, causing an expansion. (B) Hairpins or quadruplexes caused by unprocessed 5′ flaps can form during gap repair or on an Okazaki fragment, leading to expansions. (C) Misalignment of repeat units (gray blocks) during homologous recombination can lead to the addition of repeat units (expansion event) or loss of repeat units (contraction event). (D) Resection during double-strand break repair exposes repeat units that can misalign and anneal, leading to repeat unit deletion (right). Misalignment followed by slippage during gap filling could lead to an expansion (left).

Figure 3

Model for generation of repeat expansions during MMR and BER. (A) Mismatch repair (MMR) is initiated when MutSβ (a heterodimer of MSH2/MSH3) binds to and stabilizes the hairpin formed by the repetitive DNA element. PCNA loading (facilitated by RFC, not shown) also occurs at the extrusion. The interaction between MutSβ and PCNA activates the cryptic endonuclease activity of the MutLα complex (a heterodimer of MLH1/PMS2). MutLα can nick either strand; here, a nick on the top strand is shown. Exo1 exonucleolytic activity digests the end to form a single-stranded gap that extends ~150 nt past the hairpin. The hairpin is unwound by helicase activity, potentially Sgs1/WRN (see section ‘‘The role of helicases in resolving hairpins during repair and fork restart’’), and Polδ fills the gap. The nick is sealed by Lig1. These steps are repeated on the other strand to resolve the second hairpin; DNA synthesis through the hairpin at this stage leads to an expansion on both strands. Note that after nicking, repair could also proceed by strand displacement (indicated by dotted gray arrow to part B), leading to an expansion by slippage during replication or inefficient flap cleavage by Fen1. (B) BER is initiated to repair 8-oxoG lesions caused by oxidative damage. 8-oxoG is first recognized by a DNA glycosylase (Ogg1 or NEIL1). ApeI creates a nick, and strand slippage during fill in by Polβ, Polδ, Polε, or Polη can lead to an expansion (left pathway). Alternatively, hairpins can impede Fen1 cleavage (indicated by dotted Fen1), leading to inefficient or ‘‘alternative’’ flap cleavage (indicated by solid Fen1) and expansions (right pathway). A nick on the complementary (bottom) strand followed by DNA synthesis through the hairpin leads to an expansion on both strands. Alternatively, direct nicking of the hairpin, or oxidative damage within the hairpin followed by nicking, could start the BER cycle again, leading to further expansions (toxic oxidation cycle, not shown). (see colour version of this figure at www.informahealthcare.com/bmg).

Figure 4

Repeat instability through transcription-coupled repair. In this model, RNA polymerase stalls due to R loop formation and/or the formation of secondary structures on the non-template strand, which could be facilitated by an R-loop on the template strand. The folded structures could also facilitate the stall by sequestering the non-template DNA strand, thus decreasing its propensity to displace RNA (Belotserkovskii et al., 2013). An alternate model (not shown) is that the RNAPII stalls at pre-formed template hairpins (e.g. see Lin et al., 2009). Structures formed on the non-template strand during transcription could be stabilized by MutSβ binding, further increasing the strength of RNAPII stalling. Stalled transcription recruits the transcription arrest factors, including CSB and XPG, which initiate TCR. Alternatively, CSB may facilitate glycosylase activity to initiate BER, or the MutSβ complex could recruit MutLα to initiate MMR (left and right arrows). RNAPII is displaced and TFIIH is recruited; RPA and XPA stabilize the denatured bubble. The RNAPII-blocking lesion then undergoes dual incisions, the first carried out by XPF-ERCC1 that cleaves 5′ of the lesion. The second cleavage occurs downstream of the lesion and is carried out by XPG. The result would be a 25–30 nt gap that is filled by Polδ, Polε, and/or Polκ; repair replication may begin before 3′ cleavage (Staresincic et al., 2009). If the polymerase fills the gap faithfully no tract length change occurs (left). If strand slippage occurs during DNA synthesis, this leads to an expansion (center). If the polymerase replicates over a template hairpin formed due to exposed ssDNA on the bottom strand or the hairpin is excised, this will result in a contraction. (see colour version of this figure at www.informahealthcare.com/bmg).

Figure 5

Post-replicative repair at repetitive DNA elements. Repeat units indicated reflect experimental constructs, but the mechanisms described here likely work along a continuum based on repeat length. (A) Repeats that cause fork stalling or fork reversal can initiate Rad5-dependent template switching, leading to expansions. In rad5Δ cells, these expansions are eliminated. In the absence of fork stabilization proteins (Mrc1, Tof1, and Ctf18) instability and fork breakage are both increased. (B) Hairpins at medium CAG repeat tracts are bypassed during replication and Rad5-dependent template switching is initiated at post-replication gaps. Rad5-, Rad51-, Rad52-, Rad57-, and Rad54-mediated expansions occur during sister chromatid recombination. Excessive recombination is inhibited by Srs2 anti-recombinase function. (C) At short repeat lengths, Rad5-dependent PRR and unwinding by the Srs2 helicase are sufficient to fill gaps and prevent expansions without sister chromatid recombination. (see colour version of this figure at www.informahealthcare.com/bmg).

Figure 6

Helicases resolve hairpins to promote repeat stability. (A) The helicases Srs2, RTEL1, Sgs1, and WRN unwind hairpins at gaps to prevent expansions of repeat DNA. Based on instability profiles in mutants, Sgs1/WRN are hypothesized to act on the template strand to prevent contractions, and Srs2/RTEL1 on the nascent strand to prevent expansions. (B) Helicases promote replication fork progression through hairpin unwinding. Sgs1/WRN and RTEL1 may also unwind G-quadruplex structures. The placement of Srs2 at the advancing fork is based on its ability to bind the sumoylated form of PCNA, found at the replication fork, and to lessen fork stalling at CGG repeats (Anand et al., 2012). (see colour version of this figure at www.informahealthcare.com/bmg).