A Zinc Ribbon Protein in DNA Replication:  Primer Synthesis and Macromolecular Interactions by the Bacteriophage T4 Primase

Valentine, Ann M.; Ishmael, Faoud T.; Shier, Vincent K.; Benkovic, Stephen J.

doi:10.1021/bi0108554

Cited by 48 publications

(70 citation statements)

References 52 publications

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“…The surprising gain in entropy on binding for poly-G results from the unstacking of the bases upon binding to the dendrimer. Such increase in entropy of ssDNA has been observed earlier upon binding to protein 46,47 . Also the entropic and enthalpic rigidity of the polyA and polyT sequence is well known from various single molecule fluorescence studies 48 .…”

Section: Sequence Dependence Of Complexationsupporting

confidence: 72%

Structure and Dynamics of DNA−Dendrimer Complexation: Role of Counterions, Water, and Base Pair Sequence

2006

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Section: Sequence Dependence Of Complexationsupporting

confidence: 72%

Structure and Dynamics of DNA−Dendrimer Complexation: Role of Counterions, Water, and Base Pair Sequence

2006

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“…Thus, it is logical that a 6:6:1 stoichiometry exists in the active gp41⅐gp61⅐DNA complex (11). Moreover, it is likely that these rings stack concentrically in the complete primosome assembly.…”

Section: Discussionmentioning

confidence: 99%

“…The gp41 and gp61 proteins were purified by chitinbased affinity chromatography as described in ref. 11. The ssDNA substrate was 45 nucleotides in length and contained a GTT priming site, which is underlined here (5Ј-GGGTGGGA-GGGAGGT TTGCA ACTGATCGATGATAGTACGTCT-GTG-3Ј).…”

Section: Methodsmentioning

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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“…It was initially shown to accelerate the loading of 41 helicase (5). There is now evidence that 59 remains on the replication fork after loading the helicase (16, 35) 2 affects the compact structure of 32 protein covered ssDNA on the lagging strand (24), stimulates primer synthesis by the primase-helicase (36), and plays a role in coordinating leading and lagging strand synthesis (12,21,37). 59 protein has been shown to bind or cross-link to T4 41 helicase (14,18), 32 ssDNA-binding protein (5,13,14,34), DNA polymerase (35), and 61 primase (cited in Ref.…”

Section: Figmentioning

confidence: 99%

“…Loading the helicase is needed to open the duplex ahead of the leading strand polymerase and for the synthesis of primers on the lagging strand. Primer synthesis is highest in reactions with saturating levels of both 59 and 32 proteins, in addition to primase and helicase (36). Although 32 protein is not necessary for leading strand synthesis when the helicase loads (inefficiently) by itself (23), there is no leading strand synthesis in the absence of 32 protein when the helicase is loaded by 59 protein (21).…”

Section: Figmentioning

confidence: 99%

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

Jones

Green

Stephens

et al. 2004

Journal of Biological Chemistry

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Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely ␣-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys 216 and Tyr 217 , binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.Bacteriophage T4 DNA replication begins by synthesis from one of several origins in the early stage of infection, but replication from forks created on recombination intermediates becomes the predominant replication initiation mechanism at later times (1, 2). Recombination-dependent replication is made possible by the terminally redundant and circularly permuted arrangement of the 168-kb linear T4 DNA genome. Thus, the end of one molecule can invade the homologous internal region of another molecule. Phage T4 encodes the replication proteins that are needed for both modes of replication (reviewed in Refs. 3 and 4).In the T4 replication system, gene 41 helicase unwinds the duplex ahead of the leading strand T4 DNA polymerase and associates with the gene 61 primase to enable it to make the pentamer primers that initiate each lagging strand fragment. In vitro, the helicase by itself loads slowly on replication forks, but its loading is greatly stimulated by the gene 59 helicase loading protein (5). In vivo, the helicase loading protein is essential for recombination and recombination-dependent replication (1, 2). Both 41 helicase and its 59 loader are needed for polar branch migration on joint molecules formed by the T4 UvsX recombinase and gene 32 single-stranded DNA-binding protein (6), and the helicase, 59 l...

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A Zinc Ribbon Protein in DNA Replication: Primer Synthesis and Macromolecular Interactions by the Bacteriophage T4 Primase

Cited by 48 publications

References 52 publications

Structure and Dynamics of DNA−Dendrimer Complexation: Role of Counterions, Water, and Base Pair Sequence

Structure and Dynamics of DNA−Dendrimer Complexation: Role of Counterions, Water, and Base Pair Sequence

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

Mutations of Bacteriophage T4 59 Helicase Loader Defective in Binding Fork DNA and in Interactions with T4 32 Single-stranded DNA-binding Protein

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