Auxiliary ATP binding sites support DNA unwinding by RecBCD

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The RecBCD helicase is crucial for initiating double-stranded break repair in bacteria.
• RecBCD unwinds DNA at a very high rate (∼1,600 bp·s⁻¹), but the underlying mechanism remains unclear.

Purpose of the Study:

• To elucidate the mechanism behind the high processive unwinding rate of the RecBCD helicase.
• To investigate the role of ATP binding and utilization by RecBCD.

Main Methods:

• Equilibrium and time-resolved DNA binding experiments.
• Ensemble and single-molecule DNA unwinding assays.
• Crosslinking followed by mass spectrometry (XL-MS).

Main Results:

• Identified auxiliary ATP binding sites on the RecC subunit with lower affinity and distinct interactions compared to catalytic sites.
• Demonstrated the essentiality and functionality of these auxiliary sites for E. coli survival after DNA damage.
• Proposed a model where auxiliary sites enhance ATP flux to catalytic sites, optimizing unwinding rates.

Conclusions:

• Auxiliary ATP binding sites on RecC are critical for RecBCD's high-speed DNA unwinding.
• These sites ensure efficient enzyme function even under conditions of low ATP availability.
• The findings provide a new model for understanding DNA repair enzyme regulation and efficiency.