The ability of enzymes to bind to DNA and catalyze the successive addition or hydrolysis of nucleotides without dissociation is an important feature of cellular DNA replication. Polymerases and nucleases with this capability are but a subset of a diverse and poorly understood group of processive enzymes. In this project, the protein-nucleic acid interactions that result in processive hydrolysis will be studied in molecular detail via the reaction of purified exonuclease I from E. coli with specifically modified oligodeoxyribonucleotides. A characterization of this simplified system will be an important step towards being able to understand and manipulate the cellular process.
The aims of this research are the following: 1. Determine the specific sequence positions and functional groups of an oligodeoxyribonucleotide that interact with exonuclease I. 2. Determine the kinetic constants for the individual steps of the reaction. 3. Determine the conformation of an oligodeoxyribonucleotide when it is productively bound to exonuclease I. 4. Synthesize a covalent complex between exonuclease I and an oligomer and investigate the extent of intramolecular hydrolysis as a probe of the conformational requirements for processive behavior. 5. Develop a coherent model to explain the processivity of exonuclease I. The experiments will require reaction of exonuclease I with specifically modified oligodeoxyribonucleotides and determination of the kinetics and the product distributions for the reactions. Both steady state and pre-steady state measurements will be made under a variety of conditions. The conformation of the nucleic acid when it is bound to the enzyme will be determined by the spectroscopic techniques of ultraviolet absorption, circular dichroism, and fluorescence energy transfer.
