Structure-forming repeats and their impact on genome stability

Abstract

Repetitive sequences throughout the genome are a major source of endogenous DNA damage, due to the propensity of many of them to form alternative non-B DNA structures that can interfere with replication, transcription, and DNA repair. These repetitive sequences are prone to breakage (fragility) and instability (changes in repeat number). Repeat fragility and expansions are linked to several diseases, including many cancers and neurodegenerative diseases, hence the importance of understanding the mechanisms that cause genome instability and contribute to these diseases. This review focuses on recent findings of mechanisms causing repeat fragility and instability, new associations between repeat expansions and genetic diseases, and potential therapeutic options to target repeat expansions.

Figure 1.. Fork stalling at structure forming repeats results in repeat fragility and instability.

(a) Long, uninterrupted polymorphic AT repeats have the potential to form cruciform structures that serve as a barrier to replication and cause fork stalling and ATR activation. WRN (Werner Syndrome) helicase (a RecQ helicase) can be recruited to unwind the structure and prevent fork collapse. Loss of WRN results in chromosome shattering in MMR deficient MSI cancers with expanded AT repeats. (b) Pathways to resolve fork stalling at structure-forming repeats (a hairpin is shown but it could also be a G4 or triplex structure). Fork stalling can occur due to structure-forming repeats serving as a barrier to replication on either the leading or lagging strand and can result in fork reversal (a resected reversed fork is shown). Fork restart can occur through several pathways: (1) repriming past the structure, e.g. by PrimPol (2) through recombination-dependent replication (RDR), using the displaced 3’ end from a reversed fork and template strand invasion, and (3) through a BIR-like pathway after fork cleavage and end resection (referred to as broken fork repair, BFR). These pathways can result in expansions or contractions if slippage or out-of-register invasion or structure bypass occurs. Alternatively, unwinding of the structure by helicases during restart can avoid repeat instability. Exposed ssDNA accumulating during BIR can result in repeat-induced mutagenesis (RIM).

Figure 2.. Predicted models of R-loop formation at structure-forming repeats.

S-loops contain a hairpin structure formed on the non-template displaced single DNA strand opposite a DNA:RNA hybrid. G-loops contain a G-quadruplex opposite a DNA:RNA hybrid; either the displaced non-template single DNA strand can form a G-quadruplex (top) or a hybrid DNA:RNA G-quadruplex can form in the context of an R-loop (bottom). H-loops contain a triplex or H-DNA structure opposite a DNA:RNA hybrid. Nascent RNA can bind the single-stranded portion of the triplex structure and the triplex can form in two orientations, in which Hoogsteen bonding occurs either between two purine strands (top) or a purine and pyrimidine strand (bottom).

Figure 3.. Recent advances in contracting or removing expanded CAG/CTG repeat tracts.

(a) A TALEN (transcription-activator like effector nuclease) targeting expanded CAG/CTG repeats induces a DSB near the end of the repeat tract. The DSB is processed and resected by the MRX (Mre11-Rad50-Xrs2) endonuclease complex stimulated by Sae2. Repair of the gap created by end-processing occurs through single-strand annealing (SSA), resulting in contractions [70]. (b) Use of the Cas9 D10A nickase to contract or remove an expanded CAG repeat tract. Left, Cinesi et al. (2016) introduced nicks throughout the CAG repeat tract, resulting in contractions [71]. Right, Dabrowska et al. (2018) excised the CAG repeat tract by nicking outside of the tract [72]. (c) Nakamori et al. (2020) used a small molecule, NA (naphthyridine-azaquinolone) to target slipped-out CAG repeats to promote contractions [74]. NA binds only to long CAG slip-outs, for example that could form upon resolution of R-loops. Though the NA-bound CAG tract is resistant to repair, CTG hairpins on the opposite strand promote nicking or hairpin excision, and polymerase fill-in of the resulting gap will result in a bias towards contractions.