Wlodek M. Bujalowski, PhD

Professor, Department of Biochemistry & Molecular Biology

Tel: (409) 772-5634
Fax: (409) 772-1790
E-mail: wbuajalow@utmb.edu
Campus Location: 5.138D Medical Research Bldg
Mail Route: 1053


Great interest in the mechanism by which proteins interact with nucleic acids results from the extreme importance of these interactions for many vital cellular processes including replication, recombination, repair, transcription and translation. We have a long-term interest in quantitative understanding of the structure-function relationships in protein-nucleic acid interactions in solution. Such understanding can be achieved through rigorous thermodynamic, kinetic, and structural (spectroscopic) studies of both macromolecules and their relevant complexes. The current major projects in our laboratory focus on: 1.Quantitative molecular understanding of the mechanism of a replicative helicase. 2.Quantitative determination of the mechanism of DNA substrate recognition by a DNA polymerase. Both helicases and polymerases are two classes of essential enzymes involved in DNA metabolism.

Part of our work is directed toward development novel rigorous quantitative methods to study thermodynamics and kinetics of complex macromolecular interactions in solution using powerful spectroscopic techniques which include steady-state and life-time fluorescence spectroscopy, fluorescence energy transfer and anisotropy techniques, analytical ultracentrifugation, dynamic light scattering, fast chemical kinetics, and various other biochemical methods.

I. HELICASES Single-stranded DNA is a crucial intermediate in the course of DNA replication, recombination, and repair. These processes are fundamental for the transmission of genetic information. Thus, these processes require that duplex DNA is, at least transiently, unwound to form a single-stranded conformation. The unwinding reaction, possibly a rate limiting step for replication, recombination, and repair, is catalyzed by a class of enzymes called helicases. Helicases belong to a group of motor proteins which perform vectorial processes fueled by transduction of the free energy of NTP hydrolysis into a catalyzed reaction. Determination of the helicase mechanism will provide invaluable information as to how these remarkable biological machines couple the binding and hydrolysis of nucleotide triphosphates to another reaction, allowing the enzymes to perform efficient catalysis against a gradient of the chemical potential or the mechanical stress. As a primary replicative helicase in E. coli, the DnaB protein provides an outstanding model system to study the molecular mechanism of the helicase action. Our laboratory is currently examining the mechanism of the functioning of the hexameric DnaB helicase, through quantitative studies of the thermodynamics, kinetics, and structure of its complexes with nucleic acids and nucleotide cofactors. Other helicases, including the E. coli PriA protein are also quantitatively examined.

II. DNA REPAIR POLYMERASES Transmission of genetic information from one generation of cells to another, as well as repair of damaged DNA, relies on the correct replication of the cellular DNA. DNA replication is a very complex process in which the dsDNA is unwound and the two resultant single strands of the nucleic acid act as templates to guide the synthesis, one nucleotide at a time, on antiparallel primer strands. At the core of DNA replication is the nucleotidyl transfer reaction catalyzed by highly specific enzymes, DNA polymerases. Polymerase b is one of several recognized DNA-directed polymerases of the eukaryotic nucleus. The enzyme plays a very specialized function in the DNA repair machinery in mammalian cells. Pol b conducts "gapped-filling" synthesis in a processive fashion in mismatch repair, in the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in the nucleic acid. Our major interest is to elucidate the mechanism of the DNA substrate recognition by the pol b through the examination of the thermodynamics, kinetics, and structure of the enzyme complexes with the template-primer, gapped DNA, and nucleotide cofactors.


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