Forward and reverse motion of single RecBCD molecules on DNA

Area of Science:

• Molecular Biology
• Biophysics
• Enzymology

Background:

• RecBCD is a crucial enzyme involved in DNA repair and recombination.
• It possesses both helicase and nuclease activities, acting as a DNA motor.
• Understanding its precise mechanism is key to comprehending DNA metabolism.

Purpose of the Study:

• To investigate the single-molecule dynamics of RecBCD enzyme under force.
• To elucidate the step size, velocity transitions, and pausing behavior of RecBCD.
• To explore the relationship between RecBCD activity and DNA substrate conformation.

Main Methods:

• High-resolution optical trapping to apply forces up to 8 pN on individual RecBCD molecules.
• Monitoring RecBCD movement with nanometer precision to determine step size and velocity.
• Analyzing DNA elasticity changes during RecBCD translocation and backsliding.

Main Results:

• RecBCD exhibits smooth motion with a step size below 6 basepairs (bp).
• Enzyme activity is characterized by episodes of constant velocity, abrupt speed switches, and pauses (mean length 3 s).
• RecBCD can reverse direction (backslide), with DNA denaturation and reannealing observed during these events.

Conclusions:

• RecBCD-DNA complexes can exist in multiple, persistent functional states.
• These states may allow RecBCD to modulate its activity for diverse biological roles.
• The enzyme's dynamic behavior is finely tuned for efficient DNA processing.