Replication-induced DNA secondary structures drive fork uncoupling and breakage

Abstract

DNA replication is a tightly regulated, complex process that ensures the entire genome is inherited accurately in each cell division. Secondary DNA structures such as G-quadruplexes (G4s) and intercalated motifs (i-motifs) can pose a challenge to the replication machinery and in turn threaten genome stability. Multiple lines of evidence suggest G4s interfere with replication, but the underlying mechanism remains unclear. Moreover, there is a lack of evidence of how i-motifs affect the replisome. Here, we reconstituted replication of physiologically derived structure-forming sequences and found that a single G4 or i-motif is sufficient to arrest DNA replication. This ability to stall replication correlates with the stability of the secondary structure under physiological conditions. Both G4s and i-motifs trigger-helicase polymerase uncoupling, which can only be rescued by a specialised helicase, Pif1. This study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and i-motifs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.

HLTF Prevents G4 Accumulation and Promotes G4-induced Fork Slowing to Maintain Genome Stability

“…Although G4s can form in ssDNA at replication forks, 88,89,95 our data suggest that HTLF acts on G4s that form throughout the cell cycle and are associated with transcription. In vivo , HLTF’s suppression of G4s depends on its ATPase activity, although the precise mechanism is unclear.…”

“…G4 stabilization can inhibit replication fork progression, 19–21,88,89 and we previously demonstrated that HLTF promotes fork reversal and restrains fork progression in response to replication stress-inducing reagents. 54 Intriguingly, HLTF-KO cells have increased G4s and RNA-DNA hybrids in the S phase, yet they exhibit fork progression rates similar to the parental U2OS cell line.…”