A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A

Area of Science:

• Molecular Biology
• Biochemistry
• Biophysics

Background:

• Understanding DNA synthesis mechanisms at the single-molecule level is crucial for elucidating cellular processes.
• Previous studies often relied on ensemble measurements, potentially masking dynamic variations in reaction rates.
• The influence of DNA conformation on enzymatic activity remains an area of active investigation.

Purpose of the Study:

• To directly visualize and quantify DNA synthesis reactions at the single-molecule level.
• To investigate the effect of DNA conformation (stretched vs. random coiled) on DNA synthesis rates.
• To establish a method for measuring DNA synthesis reaction rates under controlled conditions.

Main Methods:

• Development of a single-stranded region tracing system using a fusion protein (RPA-YFP).
• Direct observation of DNA synthesis in microflow channels.
• Manipulation of single-stranded lambda DNA (ssλDNA) conformation using buffer flow to achieve stretched (+flow) and random coiled (-flow) states.

Main Results:

• Single-molecule DNA synthesis was successfully observed and quantified.
• The DNA synthesis rate for random coiled ssλDNA (-flow) was comparable to ensemble measurements (52 bases/s).
• Stretched ssλDNA (+flow) exhibited a 75% higher synthesis rate (91 bases/s) compared to random coiled ssλDNA, indicating DNA stretching promotes Klenow fragment activity.

Conclusions:

• The developed single-molecule observation system allows for precise measurement of DNA synthesis rates.
• DNA conformation significantly impacts DNA synthesis efficiency, with stretching enhancing the reaction rate.
• These findings provide valuable insights into the mechanical regulation of DNA synthesis and polymerase activity.