The single-stranded DNA is a crucial and active intermediate in the processes of DNA replication, recombination, and repair, which are essential for the transmission of genetic information. This intermediate is formed by a transient unwinding of the duplex DNA, which is catalyzed by a class of enzymes called helicases. The helicases are indispensable in all aspects of DNA and RNA metabolism, serving also as molecular pumps and as biological motors for large multiple-protein machines, including the replisome and pre-primosome. Genomic DNA damages, due to environmental and cellular factors, are constantly occurring in the cell, leading to the formation of the stalled replication fork. The restart of the stalled fork occurs through the assembly of the large molecular machine, the pre-primosome, which is a key process in defending the integrity of the genetic information. In E. coli, two helicases, the DnaB and PriA proteins, together with the DnaC, DnaT, PriB, and PriC proteins, are engaged in the pre-primosome assembly and functions. Elucidation of the helicase mechanisms is of paramount importance for understanding the fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases. Studying the molecular mechanisms will provide the necessary knowledge as to how to regulate and control these processes and is invaluable in designing efficient therapies for diseases. Elucidation of the mechanisms of the pre-primosome assembly and activities is crucial for understanding the rules governing the restart of the stalled fork and, in general, the functioning of large molecular machines. The E. coli DnaB protein is a paradigm model of a hexameric replicative helicase. Hexameric replicative helicases were originally classified as the DnaB-like family of enzymes. The primosome is an archetype molecular system for the collaborative action of different motor proteins and of a large molecular machine. The PriA protein is, in turn, a prototype model for the factor, which senses the presence of the stalled fork and initiates the assembly of the pre-primosome. The long-term goal of this project is to establish molecular mechanisms of replicative helicases and their engagement in the pre-primosome, and the rules, which govern the macromolecular interactions in the pre- primosome machine. This will be accomplished through quantitative studies of the thermodynamics, kinetics, and structures of multiple protein-protein and protein-DNA complexes, using the analytical ultracentrifugation, dynamic light scattering, chemical rapid quench-flow, fluorescence stopped-flow, fluorescence anisotropy, fluorescence energy transfer, crystallography, electron microscopy, and biochemical methods.

Public Health Relevance

The helicases are crucial in all aspects of DNA and RNA metabolism by catalyzing the formation of the active single-stranded intermediate and serving as motors for large molecular machines of the nucleic acid metabolism. The restart of the stalled fork is a key process in the defense of the genetic information integrity and occurs through the assembly of the pre-primosome. Elucidation of the helicase mechanisms is of paramount importance for understanding fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases, while elucidation of the mechanisms of the pre-primosome assembly and activities is crucial for understanding the rules, governing the restart of the stalled fork and, in general, the functioning of large molecular machines.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046679-20
Application #
8258780
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
1992-09-30
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
20
Fiscal Year
2012
Total Cost
$315,883
Indirect Cost
$106,688
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
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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
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
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2010) The Escherichia coli PriA helicase specifically recognizes gapped DNA substrates: effect of the two nucleotide-binding sites of the enzyme on the recognition process. J Biol Chem 285:9683-96
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2010) Interactions of the Escherichia coli primosomal PriB protein with the single-stranded DNA. Stoichiometries, intrinsic affinities, cooperativities, and base specificities. J Mol Biol 398:8-25
Kozlov, Alexander G; Jezewska, Maria J; Bujalowski, Wlodzimierz et al. (2010) Binding specificity of Escherichia coli single-stranded DNA binding protein for the chi subunit of DNA pol III holoenzyme and PriA helicase. Biochemistry 49:3555-66

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