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Time-Resolved Spectroscopic Studies of Nucleic Acid Structure and Nucleic Acid-Protein Interactions

D.P. Millar, W.C. Lam, E. Thompson, E.J.C. Van der Schans, M. Auer,* J. Burke,** C.M. Joyce,*** J.W. Orr,**** L.C. Sowers,***** N. Walter,** J.R. Williamson****

* Novartis Research Institute, Vienna, Austria
** University of Vermont, Burlington, VT
*** Yale University, New Haven, CT
**** TSRI, Department of Molecular Biology and The Skaggs Institute for Chemical Biology
***** City of Hope National Medical Center, Duarte, CA

Fluorescence spectroscopy is a powerful tool for investigating the structure and dynamics of biological macromolecules under solution conditions that mimic the intracellular environment. In our laboratory, we use picosecond time-resolved fluorescence techniques to analyze a variety of nucleic acid--protein interactions and to probe the solution structure of DNA and RNA.

Time-resolved fluorescence spectroscopy is a particularly useful technique for investigating the DNA-protein interactions involved in DNA replication and exonucleolytic proofreading by DNA polymerases. During proofreading, the 3´ end of a nascent DNA strand must shuttle from the polymerase site to the 3´-5´ exonuclease site, located in a separate structural domain of the enzyme, and the last few base pairs of the DNA must melt in readiness for the exonuclease reaction. By using DNA substrates labeled at different positions with fluorescent probes, we can directly monitor these translocation and localized melting steps. These experiments help elucidate the mechanism by which the polymerase recognizes and selectively removes misincorporated nucleotides from the nascent DNA strand.

During the past year, we investigated the structural mechanisms that enable the polymerase to correct frameshift errors during DNA replication. Frameshift errors arise through the transient misalignment of primer and template strands during replicative DNA synthesis and occur preferentially at homopolymeric and other repetitive sequences. Interest in frameshift mutagenesis has increased greatly in the past few years with the discovery that several hereditary diseases are associated with expansion of specific triplet repeat sequences in DNA. We examined the interaction between DNA polymerases and synthetic models of the misaligned DNA intermediates in frameshift mutagenesis. Our results are beginning to indicate which features of the misaligned DNA structures are recognized by the proofreading apparatus of DNA polymerases.

In collaboration with C. Joyce, Yale University, we characterized the effect of protein mutations on the partitioning of DNA substrates between the polymerase and the 3´-5´ exonuclease sites. Site-directed mutagenesis was used to change protein side chains within the polymerase domain of DNA polymerase I. From the spectroscopic analysis of these mutant enzymes, we directly quantified the energetic contribution of each side chain to the binding of DNA at the polymerase site and identified residues that distort the primer-template. These studies are providing a direct correlation between structural properties of the enzyme and the thermodynamics of the DNA-protein interactions.

We are also developing new spectroscopic techniques for the analysis of RNA structure. Time-resolved fluorescence resonance energy transfer (tr-FRET) is being used to probe the global structure and conformational flexibility of a variety of RNA molecules, including the Rev response element from HIV type 1, the hairpin ribozyme, and the central 3-way junction from 16S rRNA. The tr-FRET method provides long-range distance constraints that are used to model the overall 3-dimensional structures of these molecules. The most powerful feature of the tr-FRET method is the ability to detect and quantify different tertiary conformations of RNA. Using this approach, we monitored the conformational transition that controls the assembly of the active site in the hairpin ribozyme and dissected the interactions that stabilize the active conformation.

PUBLICATIONS

Carver, T.E., Millar, D.P. Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I. Biochemistry 37:1898, 1988.

Lam, W.C., Seifert, J.M., Amberger, F., Graf, C., Auer, M., Millar, D.P. Structural dynamics of HIV-1 Rev and its complexes with RRE and 5S RNA. Biochemistry 37:1800, 1998.

Lam, W.C., Van der Schans, E.J.C., Joyce, C.M., Millar, D.P. Effects of mutations on the partitioning of DNA substrates between the polymerase and 3´-5´ exonuclease sites of DNA polymerase I (Klenow fragment). Biochemistry 37:1513, 1998.

Lam, W.C., Van der Schans, E.J.C., Millar, D.P. Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: Implications for proofreading of frameshift errors during DNA replication. Biochemistry, in press.