Distinct quaternary states, intermediates, and autoinhibition during loading of the DnaB-replicative helicase by the phage λP helicase loader

Replicative helicases need loader proteins to assemble at DNA replication origins. Multiple copies of the bacteriophage λP (P) loader bind and load the Escherichia coli DnaB (B) replicative helicase onto single-stranded (ss) DNA from the replication origin. We find that the E. coli DnaB•λP complex exists in two forms: B6P5 and B6P6. In the 2.66 Å cryo-EM structure of B6P5, five λP loader copies form a crown-like shape that tightly grips DnaB. In this complex, the closed, planar DnaB is reconfigured into an open spiral with a large enough breach to allow ssDNA to enter an internal chamber. Transition to the open spiral involves λP-induced changes to the Docking Helix (DH)-Linker Helix (LH) interface. Unexpectedly, one λP chain in B6P5 is positioned across the breach. The disposition of this λP chain implies a complex pathway for entry of a replication-origin-derived ssDNA "bubble" ssDNA into the B6P5 complex. We propose that the B6P6 complex is an early intermediate in helicase activation in which neither DnaB nor λP has reached its final form. In this complex, DnaB adopts a partially open, ajar planar configuration. λP in B6P6 interacts more loosely with DnaB. The ssDNA- and ATP-binding sites in both complexes are not correctly configured for binding or hydrolysis. Our findings detail the distinct conformations of B6P6 and B6P5, allowing us to propose a structural model for the transition from an ajar planar to an open spiral configuration in the helicase loading pathway.