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

Abstract

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...

Mechanism of Subunit Coordination of an AAA+ Hexameric Molecular Nanomachine

“…Therefore, these data suggest that fork DNA unwinding by LT may require more than the simple translocation along ssDNA to display the other strand as described in the "exclusion model" that is currently believed to be the case for T7 gp4 and DnaB. 19,40 DNA-dependent coordination mode change for DNA unwinding As a nanomachine, LT helicase couples the energy from ATP binding/hydrolysis to DNA unwinding. Our mutant doping data using ATPase-defective or helicase-defective mutants reveal that LT unwinds DNA with clearly two distinct subunit coordination mechanisms, depending on whether a fork DNA and a blunt-ended ori-DNA serve as the substrate.…”

Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

“…The active fractions were pooled and concentrated by ultrafiltration using a YM-30 membrane. The purified helicase was greater than 98% pure as analyzed by SDS-PAGE 29, 32. The enzyme was verified to be stable for several hours at room temperature, and for at least 12 weeks at−20°C in 20% glycerol storage buffer.…”

“…The cloning and expression of the B. anthracis dnaB gene and subsequent purification of the helicase has been described 29. Briefly, the gene was amplified by PCR with primers HCASE45-5′and HCASE45-3′ (Table 6) and B. anthracis genomic DNA.…”

DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N‐terminal domain in modulating DNA binding activities

“…It has been shown by limited proteolytic digestion that a 33-kDa C-terminal fragment retains the DNA-dependent ATPase stimulation activity suggesting that this region may contain the DNA binding domain [25,26]. The ssDNA binding activity of DnaB helicases has been reported in many known helicases [12,[27][28][29][30][31]. B. stearothermophilus DnaB also binds to dsDNA, albeit with less affinity, suggesting diversity in the DNA binding activities of the DnaB helicases [12].…”

“…B. stearothermophilus DnaB also binds to dsDNA, albeit with less affinity, suggesting diversity in the DNA binding activities of the DnaB helicases [12]. Binding of ssDNA stimulates the NTPase activity of all the studied DnaB helicases [4,[27][28][29]32,33]. DnaB hexamer forms a ring-shaped structure and it is suggested that ssDNA threads through the central channel of the ring [4,31,34,35].…”