How DNA secondary structures drive replication fork instability

DNA secondary structures, such as hairpins, cruciforms, triplexes, G-quadruplexes and iMotifs, are common, dynamic features that replication forks routinely encounter. However, how these structures destabilise the replication fork remains unclear. Here, we propose a framework describing the immediate consequences of replication forks encountering DNA secondary structures. This review considers outcomes according to the affected strand (leading or lagging) and the timing of structure formation, linking strand geometry and folding dynamics to replisome behaviour. Stable, pre-formed structures on the leading strand template either impede, or are bypassed by, the CMG (CDC45-MCM-GINS) helicase, frequently leaving single-stranded DNA (ssDNA) gaps. Leading strand structures inhibit DNA polymerase ε (Pol ε), induce fork uncoupling, again producing post-replicative ssDNA gaps which can channel into fork reversal or PrimPol-dependent repriming. Lagging strand template structures inhibit DNA polymerase δ (Pol δ) and structures on 5' flaps impair Okazaki fragment maturation (OFM); both impediments yield ssDNA nicks or gaps. In each case, replication protein A (RPA) availability and the replication checkpoint define a tolerance window and coordinate hand-offs to accessory helicases, Pol δ strand displacement synthesis, and translesion synthesis (TLS). Immediate double-strand breaks (DSBs) are unlikely as an immediate consequence. Instead, we propose strand-specific ssDNA gaps predominate and may later be converted into DSBs during late S/G2 processing, mitosis, or the next S phase. This review integrates mechanisms to connect structure dynamics with fork responses and downstream ssDNA gaps and breaks, providing possible models of structure-induced genome instability.