Nucleotide and partner-protein control of bacterial replicative helicase structure and function

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Cellular DNA replication relies on hexameric helicases to unwind the DNA double helix.
• The bacterial replicative helicase DnaB is essential for initiating and elongating DNA replication forks.

Purpose of the Study:

• To elucidate the molecular mechanisms and regulatory control of the bacterial DnaB helicase.
• To investigate the structural and functional states of DnaB in response to nucleotide binding and regulatory factors.

Main Methods:

• X-ray crystallography (3.3 Å resolution) of Aquifex aeolicus DnaB.
• Electron microscopy (EM) and Small-angle X-ray scattering (SAXS) studies.
• Biochemical assays using mutant helicases with altered collar states.

Main Results:

• A novel nucleotide-bound conformational state of DnaB was identified.
• The DnaB N-terminal collar undergoes nucleotide-dependent transitions between two distinct states.
• Mutant helicases locked in specific collar states exhibit altered DNA translocation and primase interaction capabilities.

Conclusions:

• The DnaB N-terminal collar acts as an autoregulatory hub.
• This collar integrates nucleotide signals and replication factor interactions to modulate helicase activity.
• Understanding these mechanisms provides insights into the precise control of DNA replication initiation and elongation.