Structure and primase-mediated activation of a bacterial dodecameric replicative helicase.
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.
CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367, Lyon, France. Université de Lyon, F-69622, Lyon, France; Université Claude Bernard Lyon 1, F-69622, Villeurbanne, France.
Alexandre
A
Bazin
CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367, Lyon, France. Université de Lyon, F-69622, Lyon, France; Université Claude Bernard Lyon 1, F-69622, Villeurbanne, France.
Mickaël V
MV
Cherrier
Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI3265, F-38044 Grenoble Cedex 9, France.
Irina
I
Gutsche
Univ. Grenoble Alpes, Institut de Biologie Structurale, F-38044 Grenoble, France CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France CEA, Institut de Biologie Structurale, F-38044 Grenoble, France.
Joanna
J
Timmins
CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367, Lyon, France. Université de Lyon, F-69622, Lyon, France; Université Claude Bernard Lyon 1, F-69622, Villeurbanne, France laurent.terradot@ibcp.fr.
Laurent
L
Terradot
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D053488:DnaB Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D016480:Helicobacter pylori / Q000201:enzymology*; D008958:Models, Molecular; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004247:DNA; D019915:DNA Primase; D000251:Adenosine Triphosphatases; D053488:DnaB Helicases