Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Bacteriophage lambda exonuclease is crucial for DNA metabolism.
• Understanding enzyme kinetics at the single-molecule level provides detailed mechanistic insights.

Purpose of the Study:

• To investigate the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules.
• To determine the factors influencing the catalytic rate and mechanism of this enzyme.

Main Methods:

• Utilized a multiplexed approach with flow-stretched DNA to monitor single-molecule enzymatic digestion.
• Performed statistical analyses on multiple single-molecule trajectories simultaneously.

Main Results:

• The catalytic rate of lambda exonuclease is dependent on the local base content of the substrate DNA.
• DNA base melting was identified as the rate-limiting step in the catalytic cycle.
• Observed sequence-independent fluctuations in catalytic rate attributed to enzyme-DNA complex conformational changes.

Conclusions:

• The study elucidates the sequence-dependent and independent factors governing bacteriophage lambda exonuclease activity.
• DNA base melting is a critical determinant of the enzyme's catalytic efficiency.
• Enzyme-DNA complex dynamics play a significant role in enzymatic function.