Replication-induced DNA secondary structures drive fork uncoupling and breakage

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress. imageBy reconstituting eukaryotic DNA replication in vitro, this study addresses the effects of physiological quadruplex secondary structures on genome stability. G-quadruplexes (G4s) and intercalated Motifs (iMs) are found to form during replication and thereby induce replisome stalling, leading to helicase-polymerase uncoupling and nascent DNA breakage.A single physiological G4 or iM structure stalls the eukaryotic replisome by inhibiting leading strand synthesis.Helicase-polymerase uncoupling occurs following replication stalling at G4s.iMs can induce breakage on nascent DNA.Stalled forks at G4s or iMs can be rescued by the accessory helicase Pif1. In vitro reconstitution shows that a single physiological G4 or iM secondary structure stalls the eukaryotic replisome by inhibiting leading strand synthesis.image

Automated summary

This study investigates the effects of physiological quadruplex secondary structures on genome stability by reconstituting eukaryotic DNA replication in vitro. G-quadruplexes (G4s) and intercalated Motifs (iMs) are found to form during replication and thereby induce replisome stalling, leading to helicase-polymerase uncoupling and nascent DNA breakage. A single physiological G4 or iM structure stalls the eukaryotic replisome by inhibiting leading strand synthesis. Helicase-polymerase uncoupling occurs following replication stalling at G4s. iMs can induce breakage on nascent DNA. Stalled forks at G4s or iMs can be rescued by the accessory helicase Pif1. In vitro reconstitution shows that a single physiological G4 or iM secondary structure stalls the eukaryotic replisome by inhibiting leading strand synthesis.