Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation

Area of Science:

• Molecular Biology
• Biochemistry
• DNA Repair Mechanisms

Background:

• Escherichia coli RecBCD is a crucial enzyme in double-stranded DNA break repair.
• It possesses two distinct motors: RecB (3' to 5' translocase) and RecD (5' to 3' translocase).
• Existing models link DNA unwinding directly to motor translocation, but some evidence suggests uncoupling is possible.

Purpose of the Study:

• To investigate whether DNA unwinding by RecBCD can occur independently of single-stranded DNA (ssDNA) translocation by its canonical motors.
• To identify the specific RecBCD components required for this uncoupled DNA unwinding.

Main Methods:

• Designed novel DNA substrates with reverse backbone polarity linkages to stall ssDNA translocation.
• Utilized these substrates to assess RecBCD's DNA unwinding processivity and identify essential domains and activities.

Main Results:

• RecBCD demonstrated processive DNA unwinding beyond reverse polarity linkages, indicating uncoupled unwinding.
• This activity required an active RecB ATPase motor, the RecB "arm" domain, and the RecB nuclease domain (but not its catalytic activity).
• RecBCD could unwind duplex DNA for at least 80 base pairs beyond the engineered linkages.

Conclusions:

• RecBCD can unwind duplex DNA independently of ssDNA translocation by its canonical motors.
• The RecB nuclease domain plays a regulatory role in controlling RecBCD's helicase activity.
• These findings necessitate a revision of current models for RecBCD's DNA unwinding mechanism.