Structure of a helicase-helicase loader complex reveals insights into the mechanism of bacterial primosome assembly.

Bin Liu,William K Eliason,Thomas A Steitz

doi:10.1038/ncomms3495

Bin Liu, William K Eliason + Show 1 more

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https://doi.org/10.1038/ncomms3495

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Abstract

During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the ~570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed-spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release.

Highlights

During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and dissociate from the complex
In the Escherichia coli system, the helicase loader protein, DnaC, complexed with ATP, binds to hexameric helicase DnaB and forms a DnaB6–DnaC6 complex, which has been confirmed by cryo-electron microscope studies[4,5]
Primosome assembly in Grampositive bacteria is different in the details, including that the corresponding helicase is named DnaC in some bacteria such as Bacillus subtilis, and the loader protein is DnaI; the assembly of the helicase and loader protein complex onto the replication origin is assisted by a pair of co-loader proteins DnaB and DnaD in B. subtilis[10,11,12,13]

Summary

Introduction

During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and dissociate from the complex. The loader protein delivers the helicase onto the melted DNA single strands of the DnaA–oriC nucleoprotein complex at the origin of replication[2,6] In vivo, this delivery is associated with the initiator protein DnaA2,7, whose amino-terminal domain (NTD) is thought to have a role in loading the helicase and helicase loader complex onto the oriC by interacting with helicase DnaB8. After the loader protein dissociates from the helicase ring, the NTD of the helicase interacts with the carboxy-terminal domain (CTD) of the primase and forms a functional primosome that synthesizes RNA primers[9]. The more recent cryoEM structure shows a spiral arrangement of the two interacting hexamers with a split ring, consistent with the possibility of a ring-breaking role of the loader[5]

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