DnaB Drives DNA Branch Migration and Dislodges Proteins While Encircling Two DNA Strands

Kaplan, Daniel L.; O'Donnell, Mike

doi:10.1016/s1097-2765(02)00642-1

Cited by 137 publications

(147 citation statements)

References 38 publications

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“…Many helicases have been shown to move unidirectionally along nucleic acid (5,30,48) as well as to displace bonded moieties along their path without specifically interacting with these moieties (5)(6)(7)(8). Thus, unidirectional translocation is a basic activity of helicases and base pair separation might occur as a consequence of the movement.…”

Section: Discussionmentioning

confidence: 99%

“…During DNA unwinding, T7 helicase ring surrounds the 5Ј strand and excludes the 3Ј strand from its central channel (31,32). No DNA unwinding is observed when the ring helicase surrounds both strands of the DNA (6,33,34).In this paper, we study the DNA-unwinding reaction catalyzed by T7 helicase at different dTTP concentrations. We measure the single-turnover kinetics of DNA unwinding and propose a modified stepping model that allows global fitting of the unwinding kinetics to obtain the helicase's kinetic step size, stepping rate, and processivity.…”

mentioning

confidence: 99%

“…The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5)(6)(7)(8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA.…”

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confidence: 99%

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confidence: 99%

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The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong

Levin

Patel

2004

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

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Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on singlestranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp͞s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.H elicases are ubiquitous proteins that are involved in various DNA and RNA metabolic processes that require the separation of double-stranded (ds)DNA into single strands, the removal of secondary structures in RNA, or the dissociation of proteins from nucleic acids (1-4). To perform these functions, helicases use the chemical energy from NTP hydrolysis to drive the mechanical processes of translocation and nucleic acid strand separation. In this paper, we study the mechanism of DNA unwinding by bacteriophage T7 helicase that is involved in DNA replication.During replication, the helicase has to unwind a long stretch of DNA, and that requires the helicase to couple strandseparation activity to translocation. The mechanisms of these critical processes of the helicase reaction are largely unknown. It is becoming evident that helicases can move unidirectionally along nucleic acid and displace bonded moieties along their path without specifically interacting with these moieties (5-8). Thus, unidirectional translocation is a basic activity that helicases can perform without requiring interactions with the duplex DNA. Nucleic acid strand separation is a thermodynamically unfavorable process and it is made feasible by the binding of the helicase to the newly unwound strands. Numerous mechanisms of unwinding have been proposed (2-4, 9-13), but additional experimental data are needed to distinguish between these mechanisms. For the monomeric or dimeric helicases such as the Escherichia coli PcrA and Rep helicases (14-16), it has been proposed that unwinding occurs by an active mechani...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Jeong

Levin

Patel

2004

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

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“…The primase domains fan out from the helicase ring, with few self-contacts, and are connected through highly flexible linkers to the helicase domains. The expanded ring of the heptamer allows binding of dsDNA in the central channel, thereby possibly conferring a translocation activity on the heptamer (8). The flexibility of the linkage between each primase and helicase subunit allows each of the two enzymatic activities to operate nearly independently of one another yet, in the case of primase, to enjoy the increased priming activity from an oligomeric assembly versus a monomer.…”

mentioning

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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Macromolecular complexes that unwind nucleic acids

Hippel

Delagoutte

2003

BioEssays

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In this essay, we consider helicases, defined as enzymes that use the free energies of binding and hydrolysis of ATP to drive the unwinding of double-stranded nucleic acids, and ask how they function within, and are "coupled" to, the macromolecular machines of gene expression. To illustrate the principles of the integration of helicases into such machines, we consider the macromolecular complexes that direct and control DNA replication and DNA-dependent RNA transcription, and use these systems to illustrate how machines centered around coupled polymerase-helicase systems can be regulated by small changes in the interactions of their functional components.

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DnaB Drives DNA Branch Migration and Dislodges Proteins While Encircling Two DNA Strands

Cited by 137 publications

References 38 publications

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

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

Macromolecular complexes that unwind nucleic acids

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