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
Research
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.
Publications