Flexibility of the Rings: Structural Asymmetry in the DnaB Hexameric Helicase

Yang, Shixin; Xiong, Yong; VanLoock, Margaret S.; Jezewska, Maria J.; Bujalowski, Wlodzimierz; Egelman, Edward H.

doi:10.1016/s0022-2836(02)00711-8

Cited by 78 publications

(95 citation statements)

References 57 publications

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“…Unlabeled, uniformly 13 C, 15 N-labeled, and selenomethionine-substituted DnaG-C were purified as described (30). To avoid the possibility of heterogeneity at the C terminus of the protein, DnaG primase was expressed in a suppressor-free host strain (cf.…”

Section: Methodsmentioning

confidence: 99%

“…Although there is no high resolution structure reported for the intact hexameric DnaB, the structure of its smaller N-terminal domain has been determined both by NMR spectroscopy (12) and x-ray crystallography (13). These structures were recently used in conjunction with the known crystal structure of the helicase domain of the related hexameric bacteriophage T7 gp4 helicase-primase (14) to model structures into electron density maps of DnaB 6 produced by image reconstruction from negative stain electron microscopy data (15). It is generally accepted that the C-terminal face of DnaB 6 is directed into the replication fork and contains the site of strand separation, whereas the N-terminal face points away from the fork where it would be appropriately placed to interact with the DnaG primase (16,17).…”

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confidence: 99%

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Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Oakley

Loscha

Schaeffer

et al. 2005

Journal of Biological Chemistry

107

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During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB 6 . Determination of the 2.8-Å crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primasehelicase complex at replication forks.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Oakley

Loscha

Schaeffer

et al. 2005

Journal of Biological Chemistry

107

View full text Add to dashboard Cite

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“…As revealed by electron micrographs, the DnaB helicase from Escherichia coli exists as an equilibrium mixture of two structural shapes: a hexamer with sixfold symmetry and a trimer of dimers with threefold symmetry (1). The hexamers are stabilized by Mg 2ϩ ; the equilibrium between the two ring forms can be shifted by the addition of nucleotides, such as ATP␥S, which favors a threefold symmetric ring (2).…”

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confidence: 99%

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

Norcum

Warrington

Spiering

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATP␥S. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed ''open'' and ''closed.'' The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primaseto-ssDNA stoichiometry of 6:1.three-dimensional structure ͉ DNA replication ͉ protein hexamers ͉ replisome T he replicative helicases and primases are members of the primosome subassembly that is a key and integral unit in the function of the replisome during DNA replication. These proteins, isolated from diverse organisms, have been the subjects of structural studies as part of a broad effort to relate their structure to their function.The replicative helicases show common hexameric architecture. As revealed by electron micrographs, the DnaB helicase from Escherichia coli exists as an equilibrium mixture of two structural shapes: a hexamer with sixfold symmetry and a trimer of dimers with threefold symmetry (1). The hexamers are stabilized by Mg 2ϩ ; the equilibrium between the two ring forms can be shifted by the addition of nucleotides, such as ATP␥S, which favors a threefold symmetric ring (2).Key crystal structures of the active helicase domain of the phage T7 gene 4 protein revealed a hexameric structure whose subunit arrangements showed some deviation from a sixfold symmetry. The variation was ascribed to structural disruption postulated to accommodate stepwise ATP binding and hydrolysis, now reflected in this ''trapped'' structure (3). Threedimensional reconstruction and electron microscopy images of the intact T7 gp4 helicase͞primase also revealed a hexameric structure with sixfold symmetry (4). Nuclease protection and cross-linking experiments suggest that ssDNA passes through the hole in the center of the hexameric ring. Electron microscopy examination of the SV40 large T antigen (5) identified the helicase domains also as lying within a hexameric ring in an orientation identical to that seen by means of electron microscopic reconstruction of the bacteriophage T7 gp4 helicase (6).The crystal structure of the intact bifunctional helicase͞ primase from phage T7, however, revealed a heptameric ring (7). Retrospective electron microscopy showed that both hexameric and heptameric oligomeric forms were present. The heptameric ring, which contains the helicase domain, is relatively flat, but the adjoining subunits do not show true ...

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“…Previously published biochemical data suggest that the sequence-and structure-related T7 gene 4 helicase and EcDnaB make contact with the displaced strand during DNA unwinding (39 -41). Like Twinkle, EcDnaB, which also forms a ring-like structure (42,43), fully tolerates the cPu lesion in the translocating or non-translocating strands (44). The differences between cPu and Tg on DNA structure are significant.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

Khan

Crouch

Bharti

et al. 2016

Journal of Biological Chemistry

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Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5 to 3 helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5 or 3 single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5 to 3 directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

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Flexibility of the Rings: Structural Asymmetry in the DnaB Hexameric Helicase

Cited by 78 publications

References 57 publications

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Architecture of the bacteriophage T4 primosome: Electron microscopy studies of helicase (gp41) and primase (gp61)

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

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