Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• G-quadruplex (GQ) structures are DNA secondary structures formed from guanine-rich sequences.
• Specialized helicases, including RNA helicase AU (RHAU), Bloom helicase (BLM), and Werner helicase (WRN), counteract stable GQ formation in genomic DNA.
• The precise substrate specificity and unfolding mechanisms of these GQ-resolving helicases remain incompletely understood.

Purpose of the Study:

• To investigate the substrate specificity of RHAU, BLM, and WRN on G-quadruplex structures.
• To elucidate the common mechanism employed by these helicases for GQ unfolding.
• To understand how helicase activity impacts GQ ligand binding and DNA structure resolution.

Main Methods:

• Utilizing biochemical assays to assess the activity of RHAU, BLM, and WRN on various G-quadruplex conformations.
• Employing biophysical techniques to monitor the unfolding process and repetitive disruption of GQ structures.
• Investigating the effect of helicase activity on the dissociation of G-quadruplex-binding ligands.

Main Results:

• RHAU, BLM, and WRN display distinct specificities for different G-quadruplex conformations.
• All three helicases utilize a common mechanism of repetitive unfolding to disrupt GQ structures.
• This repetitive unfolding activity effectively dislodges stably bound GQ ligands, such as BRACO-19, NMM, and Phen-DC3.
• RHAU's unfolding activity promotes efficient intramolecular annealing within the same DNA molecule.

Conclusions:

• Different helicases are specialized to resolve specific G-quadruplex structures.
• A conserved repetitive unfolding mechanism underlies the robust resolving power of these helicases.
• This mechanism provides a robust strategy for managing G-quadruplex structures in biological systems.