Essential Amino Acids of Escherichia coli DnaC Protein in an N-terminal Domain Interact with DnaB Helicase

Ludlam, Anthony V.; McNatt, Matthew W.; Carr, Kevin M.; Kaguni, Jon M.

doi:10.1074/jbc.m101940200

Cited by 50 publications

(72 citation statements)

References 48 publications

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“…DnaC protein is another major component of E. coli chromosomal DNA replication (15,17). 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).…”

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

confidence: 99%

“…Binding of DnaB to singlestranded DNA (ssDNA) 1 is controlled by its interaction with DnaC. Association with DnaB stimulates the DNA binding activity of DnaC, and with the help of DnaA, it loads DnaB on the ssDNA (15)(16)(17). In the next step, primase, the DnaG gene product and a single-stranded DNA-dependent RNA polymerase, is recruited to the replication fork through a transient protein-protein interaction with DnaB.…”

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

confidence: 99%

mentioning

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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“…Because amino acid substitutions within residues 10-44 near the amino terminus of DnaC impair the interaction between DnaC and DnaB, this region of DnaC apparently binds directly to DnaB to form the DnaB -DnaC complex (Ludlam et al 2001). Initial studies showed that six DnaC monomers bind per DnaB hexamer (Wickner and Hurwitz 1975;Kobori and Kornberg 1982;Lanka and Schuster 1983).…”

Section: Dnaa Is the Replication Initiator In Bacteriamentioning

confidence: 99%

Helicase Loading at Chromosomal Origins of Replication

Bell

Kaguni

2013

Cold Spring Harbor Perspectives in Biology

116

109

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Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.

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“…The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 4 The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; SSB-Ct, SSB C terminus; ITC, isothermal titration calorimetry. …”

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confidence: 99%

Structure and Function of the PriC DNA Replication Restart Protein

Wessel

Cornilescu

et al. 2016

Journal of Biological Chemistry

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Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the singlestranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii. PriC forms a compact bundle of ␣-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB⅐DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB⅐DnaC to SSB/ssDNA sites present at stalled replication forks.Replication of circular chromosomes found in many bacteria is initiated by sequence-specific binding of the DnaA initiator protein to the origin of replication, oriC, which promotes duplex DNA melting (1-4). Single-stranded DNA (ssDNA) 4 exposed by DnaA unwinding is rapidly bound by the ssDNAbinding protein (SSB). DnaA, along with the helicase loader DnaC, then directs loading of the replicative helicase, DnaB, onto the SSB-coated ssDNA (4 -7). The remaining replication proteins are recruited through protein interactions to form the full replication complex, termed the replisome (8 -12). With each round of replication, two replisomes are loaded at oriC to replicate bidirectionally around the chromosome until converging at the terminator region (13).Replisomes assembled at oriC frequently encounter physical barriers, such as damaged DNA or genome-bound protein complexes (e.g. transcription machinery), that can stall and/or prematurely dissociate the replisome from the DNA template (14). Estimates from studies in Escherichia coli suggest that very few replisomes translocate to the replication terminus without dissociating at least once during each replication cycle (15). Because unrepaired premature termination events lead to incomplete replication, genome instability, and cell death, DNA replication restart mechanisms that reload replisomes onto abandoned replication forks are essential in bacteria (16). Due to the sporadic nature of replication failure, DNA replication restart pathway...

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Essential Amino Acids of Escherichia coli DnaC Protein in an N-terminal Domain Interact with DnaB Helicase

Abstract: Escherichia coli

Cited by 50 publications

References 48 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

Helicase Loading at Chromosomal Origins of Replication

Structure and Function of the PriC DNA Replication Restart Protein

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