Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Faithful genome duplication relies on DNA replication proteins overcoming obstacles like G-quadruplexes.
• G-quadruplexes are higher-order DNA structures that impede genomic maintenance.
• Understanding protein-DNA interactions at these structures is crucial.

Purpose of the Study:

• To develop a real-time method for observing DNA polymerase interactions with G-quadruplexes.
• To investigate how DNA polymerases behave when encountering G-quadruplex obstacles.
• To provide a versatile single-molecule assay for studying DNA-protein-obstacle interactions.

Main Methods:

• Real-time fluorescence microscopy in a microfluidic flow cell.
• Immobilization of primed DNA oligonucleotides containing G-quadruplexes on functionalized glass coverslips.
• Monitoring of fluorescently labeled DNA polymerases over time.

Main Results:

• Successfully visualized DNA polymerase stalling at G-quadruplex structures.
• Observed that yeast polymerase δ exhibits a continuous binding and unbinding cycle when encountering a G-quadruplex.
• Demonstrated the utility of the single-molecule assay for studying polymerase dynamics.

Conclusions:

• The developed method allows real-time observation of DNA polymerase behavior at G-quadruplexes.
• DNA polymerases engage in dynamic binding-unbinding cycles when stalled by G-quadruplexes.
• This assay platform can be adapted to study various DNA-maintenance proteins and DNA obstacles.