The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein

Biswas, Esther E.; Biswas, Subhasis B.; Bishop, James E.

doi:10.1021/bi00371a019

Cited by 37 publications

(51 citation statements)

References 31 publications

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“…Alternatively, 3D EM reconstructions of an Ec DnaB•DnaC complex have shown that upon binding of DnaC, contacts between the globular domains of the DnaB N-terminal collar rearrange into a cracked , but constricted, configuration (Arias-Palomo et al, 2013). Given that DnaC is present in our unwinding assays, and that the loader is known to prevent DnaB from unwinding DNA when it stays associated with the helicase (Biswas et al, 1986; Wahle et al, 1989), the observed differences in rate between wild-type and mutant DnaBs seen here could derive from a change in the relative affinity of DnaC for the helicase depending on the state of the collar. In this view, a constricted collar state would favor DnaC binding, slowing release of the loader from the helicase and leading to a long lag that precedes unwinding.…”

Section: Resultsmentioning

confidence: 81%

Nucleotide and Partner-Protein Control of Bacterial Replicative Helicase Structure and Function

et al. 2013

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Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB complexed with nucleotide reveals a new conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active, but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an auto-regulatory hub that controls the ability of the helicase to transition between different functional states in response to nucleotide and both replication initiation and elongation factors.

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Section: Resultsmentioning

confidence: 81%

Nucleotide and Partner-Protein Control of Bacterial Replicative Helicase Structure and Function

et al. 2013

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“…San Martin et al (30) has shown that the DnaB oligomer is a trimer of asymmetrical dimers with a pronounced triangular shape. In addition, DnaB exhibits three high affinity binding sites for ATP analogs, which further supports the 3-fold symmetry of this hexameric molecule (48,50). In the case of the DnaC-DnaB hexamer complex, each DnaB monomer provides a binding site for a DnaC and thus exhibits a 6:1 stoichiometric ratio (56).…”

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

confidence: 70%

“…This replication factor is required for assisting DnaB to bind to the replication origin and appears to have a pivotal role in initiation of helicase function (50,51). Previous studies demonstrated that in vitro DnaC forms a tight complex with the DnaB helicase in the complete absence of nucleotides and DNA (52).…”

Section: Analysis Of In Vitro Primer Synthesis In the Presence Of Dnamentioning

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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“…It is likely that this structural dimerization correlates with the biochemical non-equivalence of subunits observed for other hexameric helicases. The T7 gp4 hexamer, for example, has been shown to contain only three, not six, high affinity ATP-binding sites (52), as has the hexameric rho protein (53) and DnaB (54).…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Electron Microscopic Image Analysis of the Hexameric E1 Helicase

Fouts

Xiong

Egelman

et al. 1999

Journal of Biological Chemistry

109

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DNA replication initiator proteins bind site specifically to origin sites and in most cases participate in the early steps of unwinding the duplex. The papillomavirus preinitiation complex that assembles on the origin of replication is composed of proteins E1 and the activator protein E2. E2 is an ancillary factor that increases the affinity of E1 for the ori site through cooperative binding. Here we show that duplex DNA affects E1 (in the absence of E2) to assemble into an active hexameric structure. As a 10-base oligonucleotide can also induce this oligomerization, it seems likely that DNA binding allosterically induces a conformation that enhances hexamers. E1 assembles as a bi-lobed, presumably double hexameric structure on duplex DNA and can initiate bi-directional unwinding from an ori site. The DNA takes an apparent straight path through the double hexamers. Image analysis of E1 hexameric rings shows that the structures are heterogeneous and have either a 6-or 3-fold symmetry. The rings are about 40 -50 Å thick and 125 Å in diameter. The density of the central cavity appears to be a variable and we speculate that a plugged center may represent a conformational flexibility of a subdomain of the monomer, to date unreported for other hexameric helicases.The synthesis of duplex DNA is a complex enzymatic process that requires the coordination of large numbers of proteins. The mechanisms are elaborate in part because the enzymes that use the complementary template strand as a guide for nucleotide incorporation catalyze this synthesis only in the 5Ј to 3Ј direction. Given the antiparallel nature of the duplex this usually requires that two synthetic enzymes move in opposite polarities on the two strands. Synthesis of the so-called lagging strand is discontinuous and requires the cyclical association of the enzyme, while synthesis of the other strand is continuous. Nevertheless, in many prokaryote replication systems it is clear that coordination of these enzymes is achieved and maintained by a dimeric polymerase that creates a looped DNA structure in the lagging strand. This loop is mediated by multiple protein-protein interactions across the growing fork (Ref. 1, and references therein). Helicases are enzymes that can catalyze the unwinding of the template strands ahead of the fork, thus allowing for new complementary strand DNA synthesis. They were initially discovered as ancillary factors required for synthesis, but recently this view of the helicase activity has been characterized as "naive" or at least incomplete (2). Compelling evidence has been presented demonstrating that the helicase is an integral member of a large protein complex that serves as a molecular motor or pump for the replication apparatus empowering the polymerase and increasing the rate of DNA polymerase synthesis (3). The Escherichia coli dnaB helicase also plays a critical function in establishing the asymmetry at the growing fork. The helicase tracks on the lagging strand template but through interactions it holds the leading strand DN...

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The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein

Cited by 37 publications

References 31 publications

Nucleotide and Partner-Protein Control of Bacterial Replicative Helicase Structure and Function

Nucleotide and Partner-Protein Control of Bacterial Replicative Helicase Structure and Function

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

Biochemical and Electron Microscopic Image Analysis of the Hexameric E1 Helicase

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