Single-molecule DNA repair in live bacteria

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Cellular DNA damage necessitates balanced repair pathways to prevent toxic intermediate accumulation.
• In vivo organization and function of DNA repair enzymes have been challenging to study due to limitations in observing individual reactions.
• Understanding DNA repair mechanisms at the single-molecule level is crucial for cellular health.

Purpose of the Study:

• To directly visualize and quantify the in vivo dynamics of single DNA polymerase I (Pol) and ligase (Lig) molecules during DNA repair.
• To determine enzymatic rates, substrate search times, and diffusion characteristics of repair enzymes in live Escherichia coli.
• To elucidate the systems-level organization and efficiency of a model DNA repair pathway.

Main Methods:

• Utilized photoactivation, localization, and tracking techniques in live Escherichia coli.
• Visualized single fluorescently labeled DNA polymerase I (Pol) and ligase (Lig) molecules.
• Measured enzymatic rates, copy numbers, search times, diffusion, and spatial distribution of reaction sites at the single-cell level.

Main Results:

• Single DNA repair events mediated by Pol and Lig were observed to be rapid, lasting 2.1 s and 2.5 s, respectively.
• Pol and Lig activities increased significantly within minutes following DNA methylation damage.
• Enzymes spent over 80% of their time searching for substrates, minimizing the number and lifetime of toxic repair intermediates.

Conclusions:

• The study provides a quantitative, systems-level description of a model DNA repair pathway in vivo.
• Direct single-molecule observations reveal the efficiency of DNA repair enzymes in minimizing toxic intermediates.
• The developed approach offers a powerful tool for studying enzyme dynamics and reaction kinetics in living cells.