Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA.

Area of Science:

• DNA structure and metabolism
• Molecular biology
• Genomics

Background:

• G-quadruplexes (G4s) are non-canonical DNA structures crucial for DNA metabolism.
• G4s can cause replication fork stalling and genomic instability if not resolved.
• Single-stranded DNA-binding proteins and helicases are essential for resolving G4 structures.

Purpose of the Study:

• To investigate the interplay between RPA and helicases on G-quadruplexes.
• To elucidate the mechanisms by which RPA prevents, resolves, and assists helicases in eliminating G4s.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) was used to characterize the interactions.
• The study focused on human RPA, human BLM, and yeast Pif1 helicases.

Main Results:

• Human RPA efficiently prevents G4 formation by binding to single-stranded DNA (ssDNA) before folding.
• RPA interacts differentially with folded G4s, and its presence is required for stable G4 unfolding by helicases.
• Helicases like BLM and Pif1 show different G4 preferences than RPA, primarily based on loop lengths.
• RPA promotes repetitive unfolding by helicases, leading to a stable linear DNA state.
• BLM can bypass RPA-disrupted G4s and continue unwinding downstream duplex DNA.

Conclusions:

• RPA plays a multifaceted role in managing G4 structures, acting both preventatively and in facilitating their resolution.
• The combined action of RPA and helicases is crucial for the stable and efficient elimination of G4s.
• Understanding these mechanisms provides insight into maintaining genomic stability and DNA metabolism.