Communication
The HexamericE. coliDnaB Helicase can Exist in Different Quarternary States

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Abstract

The DnaB protein is the primary replicative helicase inEscherichia coli, and the active form of the protein is a hexamer. It has been reported that the protein forms a ring with strong 3-fold symmetry, which was suggested to be a trimer of dimers. We show that under different conditions, using either ATP, ATPγS, AMP-PNP or ADP as nucleotide cofactors, we always find two different forms of the DnaB ring; one with a 3-fold symmetry and one with 6-fold symmetry. We have used scanning transmission electron microscopy for mass analysis, and have found that both forms are hexamers, excluding the possiblity that the 3-fold form is in fact a trimer of the 52 kDa monomer. We have also found rings that are in an intermediate state between these two. The existence of hexamers in discrete states shows that the transitions between these states must be cooperative. These observations suggest that there may be an equilibrium between two different conformations of the hexameric ring. The role of these two states in the mechanism of helicase action remains to be determined.

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    DnaB-family proteins serve as central replicative helicases of bacteria (reviewed in [49]). Structural studies, including both EM and X-ray crystallography, have shown that DnaB orthologs readily form stable, closed hexameric rings [50–58]. In higher-resolution crystal structures, DnaB-family helicases have been found to adopt a two-tiered architecture, whereby a set of N-terminal primase interaction domains form a trimer of dimers that sits atop a pseudo-six-fold symmetric ring of RecA-type ATPase domains [53–55].

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    Upon binding of ssDNA and nucleotide, however, the active sites of DnaB have recently been shown to condense into a more uniform, compact conformation that resembles related nucleotide-bound, hexameric helicases such as the Rho transcription termination factor and T7 bacteriophage gp4 (Itsathitphaisarn et al., 2012; Singleton et al., 2000; Thomsen and Berger, 2009). Interestingly, EM experiments have indicated that conformational heterogeneity persists within DnaB even when nucleotide is present, implying that the fully liganded helicase still exhibits significant plasticity (Yang et al., 2002; Yu et al., 1996). To better define the physical consequences of nucleotide binding to DnaB, we determined the crystal structure of the full-length helicase from A. aeolicus.

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