Single-molecule analysis of PARP1-G-quadruplex interaction.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The human genome features repetitive sequences prone to forming non-canonical G-quadruplex (G4) DNA structures.
• G4s influence nucleic acid metabolism, including replication, repair, and transcription.
• Poly (ADP-ribose) polymerase 1 (PARP1), a key anticancer target, interacts with G4s and undergoes auto-PARylation, but the mechanism remains unclear.

Purpose of the Study:

• To elucidate the molecular mechanism of PARP1 interaction with G-quadruplex DNA.
• To characterize the stoichiometry and dynamics of PARP1-G4 binding.
• To investigate the functional consequences of PARP1 binding to G4s, including auto-PARylation.

Main Methods:

• Mass Photometry (MP) for studying molecular interactions.
• Single-molecule total internal reflection fluorescence microscopy (smTIRFM) for dynamic analysis.
• Biochemical assays to measure auto-PARylation and inhibitor effects.

Main Results:

• PARP1 dynamically interacts with G4s with a precise 1:1 stoichiometry.
• Binding of a single PARP1 molecule to G4-containing DNA with a primer-template junction triggers robust auto-PARylation.
• Poly (ADP-ribose) chains added reach molecular weights of several hundred kDa.
• PARP inhibitors (EB-47, Olaparib, Veliparib) exhibit differential effects on PARP1 retention on G4-DNA versus nicked DNA.

Conclusions:

• PARP1 exhibits dynamic, stoichiometric binding to G-quadruplex DNA.
• G4 structures, in conjunction with a primer-template junction, are potent activators of PARP1 auto-PARylation.
• PARP inhibitors modulate PARP1's interaction with G4-containing DNA, suggesting potential therapeutic strategies targeting this interaction.