RPA engages telomeric G-quadruplexes more effectively than CST

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• G-quadruplexes (G4s) are stable secondary structures in guanine-rich DNA.
• Telomeric DNA frequently forms G4s, posing challenges for DNA replication and maintenance.
• Protein complexes like Replication Protein A (RPA) and CTC1-STN1-TEN1 (CST) are involved in managing telomeric G4s.

Purpose of the Study:

• To investigate the binding affinities of RPA and CST for telomeric G-quadruplex structures.
• To elucidate the roles of RPA and CST in resolving G4s at telomeres.
• To understand how G4 structures affect protein-DNA interactions.

Main Methods:

• Fluorescence anisotropy equilibrium binding measurements were used to assess protein-G4 interactions.
• Various telomeric G-quadruplex topologies were tested.
• A mutagenesis strategy was employed to study RPA's DNA-binding domains.

Main Results:

• CST binding to G-rich single-stranded DNA (ssDNA) was significantly inhibited by G4 structures.
• RPA demonstrated strong binding to telomeric G4s, with affinity largely unaffected by G4 structure.
• Disruption of multiple RPA DNA-binding domains reduced its affinity for G4 ssDNA.

Conclusions:

• RPA exhibits robust binding to telomeric G-quadruplexes, suggesting a primary role in their resolution.
• CST's inability to efficiently disrupt G4s, coupled with RPA's high abundance, supports RPA's critical function.
• RPA is likely the key protein complex for resolving G4s that impede telomere replication.