A great interest in the mechanism by which proteins interact with nucleic acids results from the importance of these interactions for such vital cellular processes as DNA replication, recombination, repair, transcription, and translation. In particular replication of DNA involves a complex, highly coordinated series of reactions in which new DNA chains are initiated and elongated on each parental strand. In E. coli dnaB protein plays an essential role in these processes. The dnaB system provides an excellent model to study these vital protein-nucleic acid interactions. Elucidation of the fundamental mechanistic details of these interactions is essential to understand why such processes dysfunction in various pathological conditions, e.g., cancer and genetic diseases. Studying different steps at the molecular level should provide necessary knowledge about how to regulate and control them. This knowledge should in turn help to design efficient therapy for the diseases. Moreover, the involvement of dnaB protein in the viral DNA replication gives an opportunity to study how viruses may subvert normal regulatory mechanisms. Our overall goals are to obtain a quantitative, molecular understanding of how the E coli dnaB protein functions as a """"""""mobile replication promoter"""""""" and helicase through replication of the bacterial chromosome, as well as during viral and plasmid DNA replication. The helicase activity of dnaB protein involves unwinding of and translocation. along DNA. These are crucial and possibly rate limiting steps for replication . To understand the biosynthesis of DNA on the molecular level it is necessary to elucidate the thermodynamics of the formation and stability of the protein-DNA complexes involved. We will apply steady-state and life-time fluorescence spectroscopy, analytical ultracentrifugation, fast chemical (stop-flow) kinetic and various other biochemical and molecular biological methods to study thermodynamic, kinetic and structural aspects of the dnaB protein's interactions with nucleic acids. In the first step thermodynamic properties of the interactions will be determined, mainly using equilibrium fluorescence titrations. Following these experiments will be ATPase and DNA unwinding (helicase) activity studies. Next, the topology of the different complexes will be determined through fluorescence energy transfer, digestion protection experiments and correlated with their observed functional activities. In the final step detailed mechanistic aspects of the interactions will be studied using fluorescence stop-flow technique.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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University of Texas Medical Br Galveston
Schools of Medicine
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Bujalowski, Wlodzimierz; Jezewska, Maria J (2014) Quantitative Thermodynamic Analyses of Spectroscopic Titration Curves. J Mol Struct 1077:40-50
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2013) Energetics of the Escherichia coli DnaT protein trimerization reaction. Biochemistry 52:1858-73
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2013) The Escherichia coli primosomal DnaT protein exists in solution as a monomer-trimer equilibrium system. Biochemistry 52:1845-57
Bujalowski, Wlodek M; Jezewska, Maria J (2012) Fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow kinetics. Methods Mol Biol 875:105-33
Bujalowski, Wlodek M; Jezewska, Maria J (2012) Using structure-function constraints in FRET studies of large macromolecular complexes. Methods Mol Biol 875:135-64
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2011) Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation. J Mol Biol 411:123-42
Szymanski, Michal R; Bujalowski, Paul J; Jezewska, Maria J et al. (2011) The N-terminal domain of the Escherichia coli PriA helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain--DNA interactions and allosteric effect of the nucleotide cofactors. Biochemistry 50:9167-83
Bujalowski, Wlodzimierz; Jezewska, Maria J (2011) Macromolecular competition titration method accessing thermodynamics of the unmodified macromolecule-ligand interactions through spectroscopic titrations of fluorescent analogs. Methods Enzymol 488:17-57
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2010) The Escherichia coli PriA helicase-double-stranded DNA complex: location of the strong DNA-binding subsite on the helicase domain of the protein and the affinity control by the two nucleotide-binding sites of the enzyme. J Mol Biol 402:344-62
Updegrove, Taylor B; Correia, John J; Galletto, Roberto et al. (2010) E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain. Biochim Biophys Acta 1799:588-96

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