DNA replication, recombination, and repair are the processes fundamental for the transmission of the genetic information of the cell from one generation to the next. These processes require that duplex DNA is at least transiently unwound to form a single-stranded intermediate. The unwinding reaction is catalyzed by a class of enzymes, helicases. Knowledge of the mechanistic details of the helicase reaction is essential to our understanding of why such processes dysfunction in various diseases, e.g., cancer and human genetic diseases. Studying different steps on the molecular level should provide the necessary knowledge about how to regulate and control them. This knowledge in turn should be very useful in designing efficient therapies for diseases. The helicases are essential for all aspects of nucleic acid metabolism in which ss nucleic acid intermediates are required. Therefore, it is of fundamental importance to understand the molecular mechanism by which the helicases function in performing their activities. As the primary replicative helicase in E. coli, the DnaB protein provides an outstanding model system to study the molecular mechanism of helicase action. This research project has two major objectives. The first objective is to formulate the quantitative dynamic model of the coupling of the free energy from ATP binding and hydrolysis to be unwinding of dsDNA by the DnaB helicase. This objective can be achieved by obtaining detailed kinetics of individual steps involved in ATP hydrolysis by the DnaB helicase in its binding to ss, dsDNA, and in the unwinding of dsDNA. To achieve this objective thermodynamics and kinetics of the conformational changes of the enzyme will also be examined. The second major objective is to correlate the kinetic model of the activity of the enzyme with the structural determinants responsible for its catalysis and unidirectional tanslocation on nucleic acid. This objective can be achieved by determining the topology of interacting sites and the structure of the nucleic acid in the complex with the helicase. To achieve these goals, we will apply steady-state, lifetime fluorescence spectroscopy, fast kinetic (stop-flow, rapid quench-flow) methods, analytical ultracentrifugation and various quantitative physical and biochemical techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046679-06
Application #
2518976
Study Section
Special Emphasis Panel (ZRG3-BBCA (01))
Project Start
1992-09-30
Project End
2000-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
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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