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1.  Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase 
Biochemistry  2009;48(31):7373-7382.
DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis (B. anthracis). The mechanism of action of B. anthracis DNA primase (DnaGBA) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaGBA binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaGBA bound to the origin of bacteriophage G4 (G4ori) with approximately eight-fold higher affinity. DnaGBA was strongly stimulated (≥75-fold) by its cognate helicase, DnaBBA during RNA primer synthesis. With the G4ori ssDNA template, DnaGBA formed short (≥ 20 nucleotides) primers in the absence of DnaBBA. The presence of DnaBBA increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaBBA is thus indicative of a positive effector role for DnaBBA. By contrast, E. coli DnaB helicase (DnaBEC) did not stimulate DnaGBA and inhibited primer synthesis to near completion. This observed effect of E. coli DnaBEC is indicative of a strong negative effector role for heterologous DnaBEC. We conclude that DnaGBA is capable of interacting with DnaB proteins from both B. anthracis and E. coli; however, between DnaB proteins derived from these two organisms, only the homologous DNA helicase (DnaBBA) acted as a positive effector of primer synthesis.
PMCID: PMC2746998  PMID: 19583259
2.  Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein 
BMC Biochemistry  2012;13:10.
Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.
We have cloned and purified SSB from Bacillus anthracis (SSBBA). In the absence of DNA, at concentrations ≤100 μg/ml, SSBBA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSBBA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)70 binding suggested that SSBBA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.
Unlike other prokaryotic SSB proteins, SSBBA from Bacillus anthracis appeared to be monomeric at concentrations ≤100 μg/ml as determined by SE-HPLC. SSBBA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSBBA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSBBA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.
PMCID: PMC3464605  PMID: 22698072
Single-stranded DNA binding protein (SSB); DNA replication; Fluorescence anisotropy; ssDNA binding; Protein-DNA complex
3.  Discovery, Characterization and Comparison of Inhibitors of Bacillus anthracis and Staphylococcus aureus Replicative DNA Helicases 
Bioorganic & medicinal chemistry  2009;17(13):4466-4476.
Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC50 ≤ 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ∼5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial vs. mammalian cells (CC50/MIC >16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (Ki = 8 μM) and is rapidly bactericidal at 4× MIC.
PMCID: PMC2776654  PMID: 19477652
S. aureus; B. anthracis; helicase; high throughput screen; aminocoumarin
4.  An Essential DnaB Helicase of Bacillus anthracis: Identification, Characterization, and Mechanism of Action▿  
Journal of Bacteriology  2008;191(1):249-260.
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 (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaBBA displayed distinct enzymatic and kinetic properties. DnaBBA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ 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 DnaBBA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaBBA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaBBA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaBBA is highly efficient in DNA unwinding and its coupling to ATP consumption.
PMCID: PMC2612439  PMID: 18931108
5.  Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase α 
Nucleic Acids Research  2003;31(8):2056-2065.
Eukaryotic DNA polymerase (pol) α is a complex of four subunits. The subunits in the yeast Saccharomyces cerevisiae are: 167, 79, 62 and 48 kDa polypeptides. The p79 subunit has no known enzymatic functions, but it is essential for growth and chromosomal DNA replication. We have analyzed the interaction between the subunits of yeast pol α, particularly p167 and p79, using a yeast two-hybrid screen and deletion analysis. We have identified the interaction sites in each of these two subunits leading to p167·p79 complex formation, and correlated our results with the available genetic data. A detailed two-hybrid analysis, using the p79 gene as the bait and a yeast genomic DNA library, identified two independent groups of positive clones. One group that displayed strong positive interaction (δ1) with p79 represented a fusion of the p167 open reading frame at 3502 bp (Ile1168), and the second group, displaying weak positive interaction (δ2) with p79, had a fusion at 3697 bp (Asn1233) with the DNA-binding domain of the yeast Gal4 transcription factor. A detailed deletion analysis of the downstream region indicated the existence of two subdomains that interact with p79. Subdomain I encompasses a 65 amino acid segment between Ile1168 and Phe1232, and subdomain II is a 25 amino acid segment between Glu1259 and Leu1283. Deletion and two-hybrid interaction analysis of the p79 subunit of pol α revealed a complementary region with two subdomains: a 67 amino acid segment between Asn189 and Gln255 (I) and a 68 amino acid segment between Glu256 and Met323 (II). The p79 subdomains I and II appeared to interact with the p167 subdomains I and II, respectively. Analysis of interaction between p62 and various deletion clones of p167 did not result in an unambiguous and stable positive interaction in the two-hybrid screen between these two subunits. A strong interaction between p167 and p62 would probably require the presence of either p79 or p48 in the complex.
PMCID: PMC153739  PMID: 12682356
6.  Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins 
Nucleic Acids Research  2002;30(13):2809-2816.
The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3′ fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.
PMCID: PMC117039  PMID: 12087164

Results 1-6 (6)