Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Replicative hexameric helicases unwind DNA through steric exclusion (SE), where one strand is encircled and the other excluded.
• The steric exclusion and wrapping (SEW) model proposes that interactions with the excluded strand's exterior surface are crucial for efficient DNA unwinding.
• The archaeal *Sulfolobus solfataricus* minichromosome maintenance (SsoMCM) helicase exemplifies unwinding via the SEW model.

Purpose of the Study:

• To investigate the role of the excluded DNA strand in the unwinding mechanism of the *Escherichia coli* (Ec) DnaB helicase.
• To determine if EcDnaB utilizes a SEW unwinding mechanism similar to SsoMCM.
• To elucidate the regulatory role of electrostatic interactions with the excluded strand in DNA unwinding.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) to observe DNA unwinding dynamics.
• Site-directed mutagenesis of conserved residues on the EcDnaB exterior surface.
• Biochemical assays measuring DNA unwinding rates using DNA and morpholino substrates.

Main Results:

• EcDnaB specifically interacts with the excluded DNA strand during unwinding, consistent with the SEW model.
• Mutations of conserved, positively charged exterior residues altered interaction dynamics and significantly increased unwinding rates.
• Neutralizing the excluded strand's charge with a morpholino substrate also dramatically enhanced unwinding rates.

Conclusions:

• The findings support the SEW model for EcDnaB, expanding the understanding of hexameric helicase mechanisms.
• Electrostatic interactions between EcDnaB and the excluded DNA strand act as a regulatory mechanism for DNA unwinding activity.
• Despite differing superfamilies and polarities, both EcDnaB and SsoMCM demonstrate the importance of excluded strand interactions in DNA unwinding.