Distinct Quaternary States, Intermediates, and Autoinhibition During Loading of the DnaB-Replicative Helicase by the...

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Replicative helicases are crucial for DNA replication initiation.
• Loader proteins are required for helicase assembly at replication origins.
• Bacteriophage lambda P (P) loader interacts with E. coli DnaB (B) helicase.

Purpose of the Study:

• To elucidate the structural mechanisms of E. coli DnaB helicase loading by bacteriophage lambda P loader.
• To characterize the distinct conformational states of the DnaB-P complex.

Main Methods:

• Cryo-electron microscopy (cryo-EM) at 2.66 Å resolution.
• Structural analysis of protein-protein and protein-DNA interactions.

Main Results:

• Identified two forms of the E. coli DnaB•λP complex: B6P5 and B6P6.
• The B6P5 complex shows a closed DnaB conformation reconfigured into an open spiral, stabilized by the P loader.
• A P chain in B6P5 unexpectedly blocks ssDNA entry, suggesting a regulatory mechanism.

Conclusions:

• The B6P6 complex represents an early intermediate in helicase activation with a partially open DnaB conformation.
• Conformational changes mediated by the P loader are essential for transitioning from B6P6 to B6P5.
• These structural insights clarify the pathway for helicase recruitment to replication origins.