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

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The precise mechanisms of DNA unwinding by helicases remain incompletely understood.
• Existing models propose DNA unwinding is driven by motor translocation along single-stranded DNA (ssDNA).
• Biochemical data for E. coli RecBCD helicase challenge this model, suggesting alternative unwinding pathways.

Approach:

• Utilized stopped-flow fluorescence and single-molecule optical tweezer experiments.
• Investigated RecBCD variants: a nuclease domain deletion mutant (RecB ΔNuc CD) and a nuclease-dead mutant (RecB D1080A CD).
• Assessed rates of ssDNA translocation and double-stranded DNA (dsDNA) unwinding on various DNA substrates.

Key Points:

• RecB ΔNuc CD unwinds dsDNA slower than wild-type RecBCD, while ssDNA translocation rates are unaffected, indicating ssDNA translocation is not rate-limiting.
• The absence of the nuclease domain, not just nuclease activity, significantly impacts unwinding rates.
• RecB ΔNuc CD exhibits slower initiation and unwinding of long DNA, and increased pausing, even without chi sites.

Conclusions:

• dsDNA unwinding by RecBCD is not solely dependent on ssDNA translocation by its motors.
• The RecBCD nuclease domain likely influences DNA base pair melting rates, possibly through allosteric regulation.
• RecBCD variants lacking the nuclease domain may mimic a post-chi sequence state, offering insights into DNA repair regulation.