The response of the Eukaryotic replisome to DNA quadruplex structures

Abstract

Sequences that can form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play a range of physiological roles. However, they 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 of stalling and how this contributes to their roles in disease remains unclear. Moreover, there is a lack of evidence as to how iMs affect the replisome. In this study, I have reconstituted replication of physiologically derived structure-forming sequences to find that a single G4 or iM is sufficient to arrest DNA replication. I describe a range of approaches to attempt to detect the formation of secondary structures within the context of duplex DNA, many of which are widely used in the literature but proved unsuccessful in the context of this project. A novel technique, using solid-state nanopores to detect structures on a single molecule level, reveals that structures form as a consequence of replication. A combination of genetic and biophysical characterisation establishes that structure forming capacity is a key determinant of replisome arrest. Mechanistically, replication fork arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Stalled forks can be rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Finally, I address how the replicative helicase, CMG, responds differently to different types of DNA secondary structures, and is eventually able to bypass a pre-existing G4, iM or hairpin structure. Altogether, this study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and iMs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.