pH-controlled quaternary states of hexameric DnaB helicase

Donate, Luis-Enrique; Llorca, Óscar; Bárcena, Montserrat; Brown, Susan E.; Dixon, Nicholas E.; Carazo, José-Marı́a

doi:10.1006/jmbi.2000.4132

Cited by 25 publications

(24 citation statements)

References 51 publications

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“…13). The symmetry state varies with pH (39), and these changes are presumed to indicate significant flexibility that may be required for conformational changes that occur during translocation on DNA templates or during loading of the helicase onto DNA at origins of replication or during replication restart at stalled forks. It is clear from work on DnaB (80, 87) and the distantly VOL.…”

Section: Structures Of Dnab and Related Hexameric Helicasesmentioning

confidence: 99%

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

144

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The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process

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Section: Structures Of Dnab and Related Hexameric Helicasesmentioning

confidence: 99%

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

144

View full text Add to dashboard Cite

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“…One N-terminal region acts as a binding pocket for DnaC and primase whereas a C-terminal domain provides a DNA binding site and is also responsible for hexamerization. DnaB homohexamer can exhibit a 3-fold as well as 6-fold rotational symmetry (26,48,55). San Martin et al (30) has shown that the DnaB oligomer is a trimer of asymmetrical dimers with a pronounced triangular shape.…”

Section: Stoichiometry Of the Complex [Primase-dnab Hexamer] Ismentioning

confidence: 99%

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Mitkova

Khopde

Biswas

2003

Journal of Biological Chemistry

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Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical crosslinker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy) 3 , and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.During the last few decades, studies on the replication of phage, plasmid, and chromosomal DNA in Escherichia coli and eukaryotic cells have established an understanding of some of the basic mechanisms of DNA replication (1-4). Reconstitution of DNA replication with purified proteins has yielded great insight into the mechanism of DNA replication as well as other aspects of DNA metabolism, such as DNA repair and recombination in prokaryotic and eukaryotic cells (5-10). The replication of the E. coli chromosome requires a large number of proteins that have to work in concert in order to successfully accomplish initiation, elongation, and termination of DNA replication (2,(11)(12)(13)(14). Thus, a careful analysis of the interactions between replication factors is of critical importance for gaining further insights into the mechanism and control of the major steps of DNA replication.Upon DnaA protein activation of the origin, DnaB helicase enters the partially unwound origin. Binding of DnaB to singlestranded DNA (ssDNA) 1 is controlled by its interaction with DnaC. Association with DnaB sti...

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“…DnaB interacts with other replisomal proteins including the DnaG primase that synthesises primers for DNA synthesis, the DnaA replication initiator protein, and the 10-subunit DNA polymerase III replicase through the t subunit. 20 Although there is as yet no crystal structure of hexameric or full-length DnaB, electron microscopy (EM) studies of the DnaB 6 oligomer have been reported, [34][35][36][37] and the structure of the N-terminal domain of DnaB has been determined by nuclear magnetic resonance (NMR) spectroscopy 38 and X-ray crystallography. 39 The EM studies showed interconversion between two symmetry states (C3 and C6) of DnaB 6 dependent on pH, 35 and that binding of DnaC locks the DnaB 6 (DnaC) 6 complex into C3 symmetry.…”

mentioning

confidence: 99%

“…20 Although there is as yet no crystal structure of hexameric or full-length DnaB, electron microscopy (EM) studies of the DnaB 6 oligomer have been reported, [34][35][36][37] and the structure of the N-terminal domain of DnaB has been determined by nuclear magnetic resonance (NMR) spectroscopy 38 and X-ray crystallography. 39 The EM studies showed interconversion between two symmetry states (C3 and C6) of DnaB 6 dependent on pH, 35 and that binding of DnaC locks the DnaB 6 (DnaC) 6 complex into C3 symmetry. 36 The C-terminal domain of DnaB carries the binding sites for DNA, nucleotides and DnaC, 31 while the N-terminal domain (DnaB-N) is essential for binding to primase.…”

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confidence: 99%

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

Watt

Urathamakul

Schaeffer

et al. 2006

Rapid Comm Mass Spectrometry

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The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.

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pH-controlled quaternary states of hexameric DnaB helicase

Cited by 25 publications

References 51 publications

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

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