Structure and primase-mediated activation of a bacterial dodecameric replicative helicase.

Alexandre Bazin,Laurent Terradot,Irina Gutsche,Joanna Timmins,Mickaël V Cherrier

doi:10.1093/nar/gkv792

Abstract

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.

Highlights

DNA replication is an essential process in all organisms and consists of the faithful duplication of the genetic material to be transmitted to daughter cells
Given that no particular flexibility of the C-terminal RecA type ATPase domain (CTD) was detected in EM studies [17], we concluded that the absence of density for the CTD-ring 2 is due to rigid-body movements of individual CTDs permitted by the high crystal solvent content (70%) and the lack of crystal contact in this area
While most DnaBs characterized to date have been reported to be hexameric, we previously found that Helicobacter pylori DnaB (HpDnaB) from H. pylori was able to form double hexamers, questioning how this structure would assemble and function on replication forks

Summary

Introduction

DNA replication is an essential process in all organisms and consists of the faithful duplication of the genetic material to be transmitted to daughter cells. Replicative helicases are hexameric ring-shaped proteins that hydrolyse ATP and unwind double-stranded DNA (dsDNA), with one strand running through the central cavity of the hexamer and the other being excluded from the ring. The replicative helicase DnaB functions as a hexamer to unwind dsDNA in the 5 to 3 direction [1]. In the absence of a ligand, the NTD-ring adopts a trimer of dimers organization [4] (described as ‘dilated’ [5]), which opens a large central channel in the helicase. In the presence of nucleotides, the NTD can adopt a triskel like structure (described as ‘constricted’), a conformation compatible with interaction with the ␶ subunit of the polymerase [5]

Methods

Results

Conclusion