E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity

Area of Science:

• Biochemistry
• Molecular Biology
• DNA Replication

Background:

• The precise mechanisms of DNA unwinding by helicases remain incompletely understood.
• Existing models often link DNA unwinding to the motor activity of helicase components.

Purpose of the Study:

• To investigate the role of the nuclease domain in the DNA unwinding process mediated by E. coli RecBCD helicase.
• To determine if single-stranded DNA translocation by RecBCD motors is essential for processive DNA unwinding.

Main Methods:

• Stopped-flow fluorescence spectroscopy to measure DNA unwinding and ssDNA translocation rates.
• Single-molecule optical tweezer experiments to analyze DNA unwinding dynamics on long DNA substrates.
• Utilized RecBCD variants with deletions or mutations in the nuclease domain (RecBΔNucCD and RecBD1080ACD).

Main Results:

• Deletion of the nuclease domain significantly slowed DNA unwinding without affecting ssDNA translocation rates.
• A nuclease-dead mutant showed no significant change in unwinding or translocation rates compared to wild-type RecBCD on short DNA.
• RecBCD variants exhibited slower unwinding initiation from blunt ends, and pauses in unwinding were observed even without chi sites.

Conclusions:

• Single-stranded DNA translocation by RecBCD motors is not rate-limiting for DNA unwinding.
• The nuclease domain of RecBCD plays a crucial role in modulating the rate of DNA unwinding, potentially through allosteric mechanisms.
• RecBCD variants lacking the nuclease domain may represent a post-chi state of the enzyme.