DYNAMIC FLUORESCENCE STUDIES OF DNA/PROTEIN COMPLEXES

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Accurate replication of DNA is an essential requirement of all living organisms, and errors made in copying genetic material can result in a wide range of disorders. Although DNA is synthesized in vivo with extremely high fidelity by DNA polymerases, it is not clearly understood how this fidelity is achieved on a molecular level. The broad, long term objective of this proposal is to understand how mismatched bases resulting from polymerase errors are recognized and selectively removed the enzyme. The proposal will initially focus on the Klenow fragment of DNA Polymerase I from E. coli, which has served as a model for describing the molecular basis of DNA replication fidelity, and will subsequently be extended to polymerases from other organisms. Removal of misincorporated bases by Klenow fragment appears to involve melting and translocation of the 3' end of the DNA into a separate editing domain of the enzyme, followed by exonucleolytic removal of the mismatched base and subsequent return of DNA to the polymerization domain. During the previous period of support, a novel solution spectroscopic method was used to make a direct measurement of DNA bound to the polymerization domain and to the editing domain. It is proposed to use this method to investigate the structural features of both the DNA and enzyme that enable mismatched base pairs to be recognized. The specific aims are: 1. Test the hypothesis that melting of two or more base pairs is required for transfer of the primer terminus into the editing domain of Klenow fragment. 2. Determine whether preferential partitioning of mismatched base pairs into the editing domain is due to increased melting capacity of the DNA, disfavored binding of DNA to the polymerization domain, or a combination of these effects. 3. Identify amino acid residues involved in DNA binding and recognition of mismatched bases. 4. Measure the rate of transfer of the primer terminus between the polymerization domain and the editing domain and identify amino acid residues that facilitate transfer. 5. Determine whether Klenow fragment suppresses frameshift mutations by rejecting misaligned sequences within the polymerization domain. 6. Characterize the editing function of other DNA polymerases m order to test the generality of the principles of mismatch recognition established for Klenow fragment. Time-resolved fluorescence anisotropy decay of dansyl-labeled DNA will be used to measure the distribution of DNA termini bound to the polymerization or editing domains of Klenow fragment. The DNA and enzyme will be judiciously modified using oligonucleotide synthesis and site-directed mutagenesis techniques, respectively, and the effect on the distribution of DNA in each domain will be determined. Thermodynamic data describing binding of mismatched DNAs to each domain of the enzyme, and melting of mismatched DNA sequences, will also be obtained. Stopped-flow fluorescence techniques will be used to measure the rate of transfer of DNA between domains. The information obtained from this study will provide insight into the molecular mechanisms used to suppress mutations during DNA replication.