Distinct Quaternary States, Intermediates, and Autoinhibition During Loading of the DnaB-Replicative Helicase by the Phage {lambda}P Helicase Loader

Abstract

Replicative helicases require loader proteins for assembly at the origins of DNA replication. Multiple copies of the bacteriophage {lambda}P (P) loader bind to and load the E. coli DnaB (B) replicative helicase on replication-origin-derived single-stranded DNA. We find that the E. coli DnaB*{lambda}P complex exists in two forms: B6P5 and B6P6. In the 2.66 [A] cryo-EM model of B6P5, five copies of the {lambda}P loader assemble into a crown-like shape that tightly grips DnaB. In this complex, closed planar DnaB is reconfigured into an open spiral with a sufficiently sized breach to permit ssDNA to enter an internal chamber. The transition to the open spiral involves {lambda}P-mediated changes to the Docking Helix (DH)-Linker Helix (LH) interface. The loader directly stabilizes the open spiral. Unexpectedly, one {lambda}P chain in B6P5 is bound across the breach, precluding entry of replication-origin-derived ssDNA into DnaBs central chamber. We suggest that the B6P6 complex is an early intermediate in the helicase activation pathway wherein neither the DnaB helicase nor the {lambda}P loader has attained its final form. DnaB in this complex adopts a partially open planar configuration, termed ajar planar. The partially ordered {lambda}P loader assembly features a much looser interaction with DnaB. The ssDNA and ATP sites in both complexes are in a configuration ill-suited for binding or hydrolysis. Our work specifies the conformational changes required for the intermediate B6P6 to transition to B6P5 on the pathway to recruitment by the initiator protein complex to the replication origin. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/522210v4_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c44809org.highwire.dtl.DTLVardef@1554deeorg.highwire.dtl.DTLVardef@16f6df9org.highwire.dtl.DTLVardef@9d12dd_HPS_FORMAT_FIGEXP M_FIG C_FIG The DnaB helicase loading pathway at the phage {lambda} replication origin populates two intermediate states, distinguished by the number of {lambda}P loaders present. The DnaB ring in the B6P6 complex is planar and partially open. Although it binds six copies of the {lambda}P loader, the ajar planar state of DnaB yields inchoate interactions with the loader. During the maturation of the complex, the planar state of DnaB is reconfigured into an open spiral in the B6P5 complex, which the pentameric {lambda}P ensemble now grips tightly. This transition required the eviction of one copy of the loader. Although the breach in the DnaB open spiral is sufficiently sized for entry of ssDNA into the internal chamber, the disposition of one {lambda}P chain loader across the single breached interface in DnaB effectively blocks the path to physiological replication origin-derived ssDNA. DnaB is depicted in white/gray ribbon format under a transparent surface. The {lambda}P chains are colored orange and shades of blue. The pink cylinder represents the expected path of ssDNA through DnaB.