CMG helicase activity on G4-containing DNA templates

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• G-quadruplexes (G4s) are non-canonical DNA structures forming in G-rich genomic regions.
• G4s pose a threat to genome stability by interfering with DNA replication processes.
• The precise mechanisms by which G4s impede replication are not fully understood.

Purpose of the Study:

• To detail a methodology for investigating the impact of G-quadruplexes on CMG helicase progression.
• To provide a reproducible protocol for studying G4-helicase interactions in vitro.
• To establish a foundation for understanding how G4s stall eukaryotic replication forks.

Main Methods:

• Purification of recombinant CMG helicase from Saccharomyces cerevisiae.
• Synthesis and purification of custom DNA oligonucleotides to create controlled G4 structures.
• DNA unwinding assays utilizing prepared substrates to monitor CMG progression.

Main Results:

• The study provides a detailed protocol for DNA unwinding assays.
• The methodology allows for controlled formation and study of G4 structures in DNA substrates.
• The described methods facilitate the investigation of CMG helicase stalling by G4s.

Conclusions:

• G-quadruplexes can stall eukaryotic replication forks by blocking CMG helicase.
• The detailed methodology enables robust investigation of G4-CMG interactions.
• This approach offers a adaptable template for studying other helicases and G4 structures.