Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Abundant DNA secondary structures, including G-quadruplexes (G4s) and intercalated-Motifs (iMs), are prevalent in the human genome.
• These structures play physiological roles but can also impede DNA replication and compromise genome stability.
• The precise mechanisms by which G4s and iMs interfere with replication remain incompletely understood.

Purpose of the Study:

• To investigate how G-quadruplexes (G4s) and intercalated-Motifs (iMs) affect DNA replication.
• To elucidate the mechanistic basis of replication arrest induced by these DNA structures.
• To identify factors involved in the resolution of replication forks stalled by G4s and iMs.

Main Methods:

• Reconstitution of DNA replication using physiologically relevant structure-forming sequences.
• Single-molecule structure detection in solid-state nanopores.
• Combined genetic and biophysical characterization of structure stability and formation probability.
• Analysis of helicase activity in resolving stalled replication forks.

Main Results:

• A single G4 or iM is sufficient to arrest DNA replication.
• These structures form during replication, as detected by nanopore analysis.
• Replication arrest results from impaired synthesis and helicase-polymerase uncoupling; iMs also cause nascent DNA breakage.
• Only the Pif1 helicase, not Rrm3, Sgs1, Chl1, or Hrq1, can rescue stalled forks.

Conclusions:

• G4s and iMs are endogenous sources of replication stress, directly inhibiting DNA replication.
• Structure stability and formation probability are critical determinants of replication fork arrest.
• Pif1 is a key helicase for resolving replication-associated G4 and iM structures.