Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC

Martı́n, C.P.; Radermacher, Michael; Wolpensinger, Bettina; Engel, Andréas; Miles, Caroline S.; Dixon, Nicholas E.; Carazo, José Marı́a

doi:10.1016/s0969-2126(98)00051-3

Cited by 71 publications

(73 citation statements)

References 39 publications

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“…Cryoelectron microscopy studies indicated that DnaC forms a hexameric ring associated with the hexameric DnaB ring (16). By analogy, we predict a similar oligomeric state and arrangement of gp59 and gp41.…”

mentioning

confidence: 64%

“…Cryoelectron microscopy studies on the E. coli DnaB and DnaC proteins, the bacterial homologs of T4 gp41 and gp59, respectively, show that the two proteins form stacked hexameric rings (16). Formation of rings is a feature conserved in DNA replication because of the thermodynamic stability of the ring as well as the ability to circumscribe DNA and form stable protein-DNA complexes.…”

Section: Resultsmentioning

confidence: 99%

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Assembly of the Bacteriophage T4 Helicase

Ishmael

Alley²,

Benkovic

2002

Journal of Biological Chemistry

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The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 singlestranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.

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mentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Assembly of the Bacteriophage T4 Helicase

Ishmael

Alley²,

Benkovic

2002

Journal of Biological Chemistry

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“…Kinetic measurements of the ATP-dependent DNA unwinding by gp41 showed maximal activity when gp59 and gp41 were present in a 1:1 mole ratio (hexamer of gp41:hexamer of gp59) (15), reminiscent of the complex of hexameric DnaC (analogous to gp59) and hexameric DnaB helicase in E. coli (16). The gp61 protein, in turn, reaches a maximal priming rate in a 1:1 mole ratio with gp41 and forms higher oligomeric complexes in the presence of DNA, implying that the active gp61 protein is also a hexamer, possibly with a ring-like structure (17).…”

mentioning

confidence: 99%

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

Zhang

Spiering

Trakselis

et al. 2005

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

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Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM.

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“…5d. These results provide evidence for gp59 forming a hexameric complex on the replication fork and loading the gp41 hexamer at a 1:1 stoichiometry (16,17).…”

Section: Discussionmentioning

confidence: 66%

“…In the analogous Escherichia coli proteins, DnaC (helicase loader) binds to the DnaB (helicase) hexamer likewise at a 1:1 stoichiometry (17). Cross-linking experiments observed complexes of gp59 up to a pentamer in the presence of gp32, gp41, and DNA.…”

mentioning

confidence: 99%

Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59)

Arumugam

Lee

Benkovic

2009

Journal of Biological Chemistry

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The T4 bacteriophage helicase loader (gp59) is one of the main eight proteins that play an active role in the replisome. gp59 is a small protein (26 kDa) that exists as a monomer in solution and in the crystal. It binds preferentially to forked DNA and interacts directly with the T4 helicase (gp41), singlestranded DNA-binding protein (gp32), and polymerase (gp43). However, the stoichiometry and structure of the functional form are not very well understood. There is experimental evidence for a hexameric structure for the helicase (gp41) and the primase (gp61), inferring that the gp59 structure might also be hexameric. Various experimental approaches, including gel shift, fluorescence anisotropy, light scattering, and fluorescence correlation spectroscopy, have not provided a clearer understanding of the stoichiometry. In this study, we employed single-molecule photobleaching (smPB) experiments to elucidate the stoichiometry of gp59 on a forked DNA and to investigate its interaction with other proteins forming the primosome complex. smPB studies were performed with Alexa 555-labeled gp59 proteins and a forked DNA substrate. Co-localization experiments were performed using Cy5-labeled forked DNA and Alexa 555-labeled gp59 in the presence and absence of gp32 and gp41 proteins. A systematic study of smPB experiments and subsequent data analysis using a simple model indicated that gp59 on the forked DNA forms a hexamer. In addition, the presence of gp32 and gp41 proteins increases the stability of the gp59 complex, emphasizing their functional role in T4 DNA replication machinery.T4 bacteriophage is one of the major model systems for acquiring information on DNA replication and repair processes. The T4 replisome is composed of eight major proteins that are subgrouped into the holoenzyme (gp43 polymerase, gp45 clamp, and gp44/62 clamp loader) and the primosome (gp41 helicase, gp59 helicase loader, gp32 single-stranded DNA-binding protein, and gp41 primase). Replication takes place through the coordinated function of all these proteins (1).The helicase loader (gp59) functions to load the helicase onto a DNA strand that is coated with gp32. In addition, gp59 inhibits the initiation of polymerization by gp43 (2, 3). Furthermore, it acts in recombination and recombination-dependent DNA replication (4, 5) and functions as a gatekeeper in origin-dependent replication in vivo (6). The gp59 protein is known to exist as a monomer in solution and in an x-ray crystal structure (7,8). It binds to several types of DNA substrates, preferring a replication fork (i.e. forked DNA) (9). Based on its similarity to the members of the high mobility group (HMG) family of proteins, a model structure has been proposed by Mueser et al. (8) for gp59 bound to forked DNA in which the N-terminal domain of gp59 binds to the duplex DNA, whereas the lagging strand passes through a narrow groove that lies between the N-and C-terminal domains and the leading strand binds to the bottom surface of the C-terminal domain.An interaction between gp59 and g...

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Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC

Cited by 71 publications

References 39 publications

Assembly of the Bacteriophage T4 Helicase

Assembly of the Bacteriophage T4 Helicase

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

Investigation of Stoichiometry of T4 Bacteriophage Helicase Loader Protein (gp59)

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