title: Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein.
abstract: DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA(BA)). DnaA(BA) was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA(BA) bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K(D) ranged from 3.8 × 10(-8) M for a specific DnaA box sequence to 4.1 × 10(-7) M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA(BA)·DNA complex formation. DnaA(BA) had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V(max)) of ATP hydrolysis without any significant change in ATP binding affinity.
authors.affiliation: Department of Molecular Biology, Graduate School of Biomedical Sciences, University of Medicine & Dentistry of New Jersey, 2 Medical Center Drive, Stratford, NJ 08084, USA.
authors.forename: Shawna M
authors.initials: SM
authors.lastname: Rotoli
authors.forename: Esther
authors.initials: E
authors.lastname: Biswas-Fiss
authors.forename: Subhasis B
authors.initials: SB
authors.lastname: Biswas
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000465:Algorithms; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D018741:Replication Origin / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate