Mechanisms of trinucleotide repeat instability during human development

Abstract

Trinucleotide expansion underlies several human diseases. Expansion occurs during multiple stages of human development in different cell types, and is sensitive to the gender of the parent who transmits the repeats. Repair and replication models for expansions have been described, but we do not know whether the pathway involved is the same under all conditions and for all repeat tract lengths, which differ among diseases. Currently, researchers rely on bacteria, yeast and mice to study expansion, but these models differ substantially from humans. We need now to connect the dots among human genetics, pathway biochemistry and the appropriate model systems to understand the mechanism of expansion as it occurs in human disease.

Figure 1. Human germ cell development

Primordial germ cell (PGC) precursors emerge during embryogenesis at day 18–19 in humans (day 7.25 in mice) as a cluster of about 20 cells. They migrate to the gonad between day 28 and 36 (or at day 10.5 in mice). Sex differentiation to spermatogonium in males and oogonium in females occurs around day 44–49 (day 13.5 in mice). In the testes of the male (top panels), the spermatogonia proliferate (curved arrow). They enter meiosis I (MI), differentiate to primary spermatocytes and undergo two meiotic reductions (MI and MII): first to secondary spermatocytes and then to haploid spermatids. The germ cells differentiate to mature sperm in the epididymus (not shown). Small expansions of pre-mutation alleles in coding and non-coding trinucleotide repeats (TNRs) are observed in both primary spermatocytes and in haploid germ cells. Large deletions of full-mutation-length alleles of non-coding TNRs are observed in dividing spermatogonia. In females (lower panels), PGCs undergo a limited number of divisions (~20) (curved arrow) and differentiate into primary oocytes that arrest during MI. The primary oocytes remain arrested in MI for years in humans. Primary oocytes complete MI at sexual maturity and form a secondary oocyte, which initiates MII but does not complete it until fertilization. Large non-coding TNR expansions occur in primary oocytes that are arrested during MI.

Figure 2. Loops formed during base excision repair by strand displacement: the toxic oxidation cycle

In base excision repair (BER), 7,8-dihydro-8-oxoguanine DNA glycosylase (OGG1) (red oval) recognizes and removes oxidized guanines (O = G) in the DNA template (steps 1 and 2). OGG1 has two enzymatic functions: glycosylase activity and lyase activity. The glycosylase activity cleaves the C1 glycosidic bond between the ribose sugar and the base. Removal of the oxidized guanine creates an apurinic site in which the widowed cytosine (C) has no partner. OGG1 can nick the phosphodiester backbone between the C3 bond of the ribose ring and the phosphate on the 3′ side of the apurinic site. The resulting single-strand break (step 2) leaves behind a residual ribose phosphate (+RP) group. The RP is not recognized by most the polymerases. However, apurinic/apyrimidinic endonuclease 1 (APE1, also known as APEX1) (dark pink oval) processes and removes the RP site and produces a 3′ hydroxyl group (OH) suitable for extension by a DNA polymerase (Pol) (purple oval). The RP site can also be removed by Pol β, which has its own lyase activity. The trinucleotide repeat (TNR) strand is displaced during gap-filling synthesis (step 3) and TNRs from the displaced ‘flap’ (step 4) can fold back into a hairpin (a flap containing CAG is shown as an example). In normal BER, flap endonuclease 1 (FEN1) excises the flap. However, the folded 5′ end of the stable TNR hairpin prevents cleavage by FEN1 (T bar). Binding of the mismatch repair recognition complex MutS homologue 2 (MSH2)–MSH3 (light pink and blue ovals) to the A-A mismatched bases (red circle in hairpin stem) may further stabilize the repetitive hairpin (step 5) and contribute to the prevention of flap removal. The hairpin DNA is ligated (step 6) and expansion occurs after the DNA hairpin loop is incorporated into duplex DNA (step 7). Base oxidation occurs daily and throughout life. Thus, continual rounds of oxidation–excision–expansion form a ‘toxic oxidation cycle’ and lead to TNR growth and progressive somatic mutation with age (step 8). If two oxidized bases occur on opposite strands, the hairpin on one strand may be copied by the polymerase into duplex DNA during gap-filling synthesis on the strand opposite the hairpin (not shown).

Figure 3. Nucleotide excision repair and trinucleotide repeat loop formation

Nucleotide excision repair has two branches: transcription-coupled repair (TCR) (left) and global genome repair (GGR) (right). In a model for the involvement of TCR in trinucleotide repeat (TNR) loop formation, the GC-rich TNR sequences block (orange star) progression of RNA polymerase II (RNAPII) (the red line represents mRNA). TCR rescues the stalled polymerase through the combined action of a group of accessory proteins (blue), including Cockayne syndrome protein CSB (also known as ERCC6). See ref. for further details. Xeroderma pigmentosum complementation group A (XPA) stabilizes the transcription bubble. The TNR tract should eventually be removed from DNA by the action of two nucleases: the XPF–ERCC1 complex (composed of XPF and excision cross complementing repair 1 (ERCC1)) and the endonuclease XPG. However, the incision by XPF–ERCC1 is made first. XPG has been implicated in TNR instability; it both binds and stabilizes the open transcription ‘bubble’. XPG is recruited to the opened DNA together with TFIIH (a transcription factor complex associated with RNAPII). Helicase and ATPase activities within TFIIH stimulate further opening of the bubble. Replication protein A (RPA) and XPA protect the ssDNA in the denatured bubble and stabilize the complex. Gap-filling repair replication (involving DNA polymerase (Pol) and associated proteins) and strand displacement from the 5′ side incision begin before the 3′ side incision is made by XPG. Thus, the transient flap created during strand displacement repair can fold back to form a stable hairpin loop at TNRs (mismatched bases in the stem of the hairpin are indicated by a red circle). In principle, GGR could also contribute to expansion, but it is likely to operate only if a looped-out structure is already formed (structural block indicated by an orange star) and constitutes a bulky lesion. However, neither of the two lesion sensing complexes of GGR has been implicated in expansion: XPC (part of the XPC–RAD23B–centrin 2 (CEN2) complex) has no effect on expansion in mice. The second sensor — a complex of DNA damage binding protein 1 (DDB1) and DDB2 — has not yet been tested for its effects on expansion. Figure is modified from ref. © (2008) Macmillan Publishers Ltd. All rights reserved.

Figure 4. Slippage model for small trinucleotide repeat length changes

Illustrated is a slippage error on the leading strand template. a | Before reaching the CAG repeat sequence (coloured bars), the replicative DNA polymerase (Pol ε on the leading strand) and DNA helicase are tightly coupled and coordinate with each other at the replication fork. b | When the polymerase encounters the CAG tract, polymerase progression is slowed but the helicase speed is unaffected (space between green and yellow ovals). c | To avoid uncoupling of the polymerase and the helicase, the leading strand polymerase bypasses a segment of unreplicated CAG template on the leading strand. d | Coupling stress is relieved when the fork has moved through the CAG repeat region and resumes copying random DNA sequence. Base pairing and processing of the looped strand results in instability of the CAG tract (not shown). Deletions occur if the loops form on the template strand and insertions occur when loop formation is on the daughter strand (not shown). Slippage can occur in both leading and lagging strand orientation, although instability (most often involving deletions) is highest when the repeats are on the lagging strand template (Pol α is the primase that synthesizes primers on the lagging strand). Figure is modified, with permission, from ref. © (2008) The American Society for Biochemistry and Molecular Biology.

Figure 5. A model for loop formation based on polymerase stalling and restarting within a trinucleotide repeat

A | Bypass of a lesion. Aa | The trinucleotide repeat (TNR) tract prevents polymerase passage on the leading strand template and it stalls (black circle), but the lagging strand can continue synthesis (green line). Ab | To overcome the block, the fork ‘backs-up’, forming a four-way junction resembling a ‘chicken foot’. Ac | The leading strand polymerase uses the newly synthesized daughter on the lagging strand as a template to synthesize enough DNA to pass the long non-coding TNR tract block (dashed line). Ad | TNR loops can occur during replication fork reversal or restart. In this example, a hairpin within the TNR tract (in green) is shown on the daughter strand of the lagging strand template, which is trapped to form an expansion intermediate. The template strand is in red; the nascent strand is in black. B | Multiple restart attempts by DNA polymerase (Pol) (purple oval) may induce large single-strand loops (second panel from left), which signal an SOS response. The SOS response induces expression of nucleotide excision repair machinery and translesion polymerases (TLPs), which restart replication. A CNG (N = C, A, G) TNR tract or a structural intermediate can be bypassed via a poorly understood ‘switching’ mechanism. A TLP (yellow oval) replaces a stalled replicative polymerase until lesion is bypassed. In a second switch, the TLP is displaced by the replicative polymerase and synthesis can resume. The TNR loops that are left behind form hydrogen bonded intermediates (hairpin with red circle). Grey arrows represent polymerase progress; the grey T bar represents polymerase stalling.

Figure 6. Three models for loop incorporation into duplex DNA

Normally, mismatch repair (MMR) machinery removes small loops in DNA (red) during replication without mutation (top panels, ‘no mutation’). However, failure to remove the loops (‘inhibition of repair’) can result in expansion. In dividing cells, the uncorrected loop (shown here as a hairpin with an A mismatch (red circle)) is copied into DNA during the next round of replication. Therefore the trinucleotide repeat (TNR) sequence increases by the size of the DNA (red ‘ladder’). In non-dividing cells, loop incorporation is not coordinated with the replication fork. The mechanism of de novo loop removal is unknown. However, two models for loop incorporation have been proposed, both of which involve a single-strand break (nicking) on the strand opposite the hairpin (grey arrow). In the canonical MMR model (centre panels), the hairpin recruits catalytically active MutS homologue 2 (MSH2)–MSH3. The MMR machinery then introduces a nick on the opposite strand from the hairpin and a polymerase (purple oval) incorporates the loop after gap-filling synthesis. In an alternative model (right panels) MSH2–MSH3 binds to the mispaired bases in the stem of the hairpin. The interaction with the hairpin alters the function of MSH2–MSH3 such that it does not successfully carry out canonical MMR. Instead, it acts as adaptor to recruit non-MMR machinery to complete loop incorporation through a crosstalk pathway.