Structure and function of Escherichia coli DnaB protein: Role of the N-terminal domain in helicase activity

Biswas, Subhasis B.; Chen, Pei-Hua; Biswas, Esther E.

doi:10.1021/bi00203a028

Cited by 44 publications

(69 citation statements)

References 34 publications

(52 reference statements)

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“…Very little is known about which amino acid residues or subdomains in DnaB are involved in these interactions. Domain ␤ (amino acids 157 to 300) contains the ATPase activity (2,24). The ATPase is characterized by two major motifs, a Walker A motif and a Walker B motif, based on comparative analysis of known ATPases (20,37).…”

Section: Resultsmentioning

confidence: 99%

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

Rosario

Singer

2007

J Bacteriol

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Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB A116V , was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

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Section: Resultsmentioning

confidence: 99%

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

Rosario

Singer

2007

J Bacteriol

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“…Partial trypsin digestion produces two domains from native DnaB, an N-terminal domain (residues 15 to 125 or 15 to 127) and a C-terminal domain (residues 170 to 470) (23). The isolated C-terminal domain contains the site for ATP binding and hydrolysis and is the location of an amino acid substitution that eliminates detectable hydrolysis (7,23,29). Other activities of DnaB, including primase stimulation, are lost upon trypsin digestion.…”

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confidence: 99%

Defect in general priming conferred by linker region mutants of Escherichia coli dnaB

Stordal

Maurer

1996

J Bacteriol

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The dnaB gene of Escherichia coli encodes a bifunctional primase accessory protein/helicase necessary for chromosomal replication. Monomers of DnaB comprise two trypsin-resistant domains connected by a 45-amino-acid linker. To investigate the role of the linker in the structure and function of DnaB, we have purified and characterized three DnaB mutant proteins having single amino acid substitutions in the linker. We find that the mutant proteins retain the two-domain structure and assemble into hexamers that may be less stable than hexamers formed by wild-type DnaB. These mutant hexamers have hydrodynamic properties slightly different from those of the wild type, suggestive of a more open structure. The mutant proteins had reduced or absent ability to stimulate primase and also exhibited slight alterations in ATPase activity compared with the wild type. We conclude that the linker region promotes primase-DnaB interaction, but this effect may be indirect. We propose a model involving repositioning of N-terminal domains to explain the properties of the mutant proteins.

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“…ATPase assays were carried out based on previously described methods (16). The amount of enzyme used in the assays was selected such that the rate of hydrolysis would be linear in the time range examined.…”

Section: Vol 191 2009mentioning

confidence: 99%

“…These domains appear to be present in all bacterial replicative DNA helicases (10,16). Previous studies involving functional analysis of DnaB EC indicate that domain ␤ contains both the ATP binding and the ATPase active sites, and domain ␥ likely includes the ssDNA binding site and one of the two sites for hexamer formation.…”

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confidence: 98%

An Essential DnaB Helicase ofBacillus anthracis: Identification, Characterization, and Mechanism of Action

et al. 2009

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We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB BA ) is a 453-aminoacid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB BA displayed distinct enzymatic and kinetic properties. DnaB BA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 533 DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB BA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB BA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB BA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB BA is highly efficient in DNA unwinding and its coupling to ATP consumption.Bacillus anthracis is a gram-positive bacterium and the etiological agent of the disease anthrax (29, 30). B. anthracis and other gram-positive bacteria pose a serious threats to human health. Thus, considerable efforts have been placed on understanding the physiology and pathology of these microorganisms. Currently, the molecular and cellular biology of B. anthracis is poorly understood. A detailed understanding of proteins involved in fundamental cellular processes such as DNA replication is critical to combating diseases caused by this organism.Chromosomal DNA replication requires the concerted actions of many different proteins in stable and transient complexes (35). Extensive studies of the process chromosomal DNA replication in Escherichia coli, as well as its plasmids and phages, have led to it serving as a model system for the study of DNA replication in both prokaryotes and eukaryotes (1, 2, 7, 15, 18, 19, 23-25, 35, 40, 41, 44, 45). DnaB protein appears to be involved in all stages of DNA replication from initiation to termination (7,21,23). The DnaB helicase is a multifunctional enzyme that is involved in the formation and translocation of the replication machinery in E. coli and bacteriophage and thus plays a pivotal role (4,6,9,11,15,22,35,38,40,42). Due to its ability to physically interact with a variety of other replication proteins, the DnaB protein plays a key role in the assembly of the primosome and subsequent movement of the replication apparatus (2,3,5,10,14,19,20,22,26,37,37,40,43). DnaB unwinds the DNA duplex into two single parental DNA template strands, which are protected by single-stranded DNA (ssDNA) binding protein (SSB). The process proceeds unidirectionally (5Ј33Ј) in a forklike manner (12, 38). Replication of the "leading strand" by DNA polymerase III holoenzyme, consisting of DNA polymerase III core and associated proteins...

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Structure and function of Escherichia coli DnaB protein: Role of the N-terminal domain in helicase activity

Cited by 44 publications

References 34 publications

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

Defect in general priming conferred by linker region mutants of Escherichia coli dnaB

An Essential DnaB Helicase ofBacillus anthracis: Identification, Characterization, and Mechanism of Action

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