Identification of a Region of Escherichia coli DnaB Required for Functional Interaction with DnaG at the Replication Fork

Chang, P.-Z.; Marians, Kenneth J.

doi:10.1074/jbc.m001800200

Cited by 40 publications

(55 citation statements)

References 44 publications

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“…This result is due to the fact that mutations R324A and R326B do not abolish, but rather significantly reduce, DNA binding and DNA-dependent ATPase and helicase activity (46). It has been shown that the DNA binding motif, RSRARR, is located in the C-terminal domain of E. coli helicase (45), whereas the primase binding site is positioned in the N-terminal domain (35). Consequently, DnaBMut1 and DnaBMut2 mutants should be able to bind primase, even though they are defective in DNA binding.…”

Section: Dnab Helicase Binding To Dna Is Necessary For Directing Thementioning

confidence: 86%

“…4B, c). Although direct interaction of these two replication proteins has been reported (39), Chang and Marians (35) have suggested that this interaction is very weak and hence, very difficult to study quantitatively. It is possible that while passing through the gel filtration column under high pressure, this complex dissociates because of its weak nature.…”

Section: Analysis Of In Vitro Primer Synthesis Carried Out By Primasementioning

confidence: 99%

“…Mutation and deletion studies have demonstrated that the N-terminal domain of primase and the C-terminal region of of DnaB are involved in the DNA binding activity (32). More precise studies reported that 16 amino acids at the C-terminal domain of primase are crucial for interaction with DnaB (33), whereas an N-terminal domain of about 12 kDa of DnaB provides the binding site for both primase and DnaC (34,35).In the E. coli replication fork, cyclic association and dissoci-* This work was supported by a grant from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

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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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Section: Dnab Helicase Binding To Dna Is Necessary For Directing Thementioning

confidence: 86%

Section: Analysis Of In Vitro Primer Synthesis Carried Out By Primasementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…As determined originally by NMR studies of larger N-terminal fragments of E. coli DnaB, the folded portion of DnaB-N comprises residues 24 -136 (48), and some mutations within this domain have been observed to affect the interaction between DnaB 6 and primase (49). However, by itself, DnaB-N-(24 -136) is an unstable protein that is partly unfolded at 37°C (50), which probably means that the structure of this part of DnaB 6 is very easy to destabilize by mutagenesis.…”

Section: Dnag-c Contains All Determinants For Helicase Binding-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

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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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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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Identification of a Region of Escherichia coli DnaB Required for Functional Interaction with DnaG at the Replication Fork

Cited by 40 publications

References 44 publications

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

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

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

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

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