STRUCTURES, INTERACTIONS AND DYNAMICS OF NUCLEIC ACIDS

University Of Missouri Kansas City, Kansas City MO

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Abstract

The central objective of this research program is to understand mechanisms of nucleic acid recognition in biological macromolecular assemblies. Emphasis is given to determining conformations and interactions of DNA in specific protein/DNA complexes that are relevant to gene regulation and chromosome organization. The methodology involves use of static and dynamic Raman spectroscopic probes of DNA structure and interaction. The specific aims of the proposed research are as follows: (1) To determine sites of DNA base and backbone interactions and local and global DNA conformations in complexes of DNA with transcriptional activators and telomere binding proteins. (2) To extend and refine Raman spectral correlations for determining nucleic acid structures in nucleoprotein assemblies and condensed states of DNA, with emphasis on issues of DNA bending and folding. (3) To extend the time-resolution of Raman dynamic probes and the concentration range of quantitative Raman assays developed previously, thus enabling novel investigations of protein/DNA recognition. These aims will be pursued using recently designed Raman approaches, including Raman microdialysis flow-cell technology, near-infrared/visible Raman spectrophotometry for ultrasensitive difference spectroscopy, Raman microspectroscopy, and ultraviolet-resonance Raman (UVRR) spectroscopy. Targeted for study are eukaryotic transcriptional activators (hSRY, LEF-1, bZIP protein GCN4) and prokaryotic factors (HU protein) bound to appropriate DNA sequences, telomere binding proteins in complexes with telomeric DNA, packaged viral chromosomes, and custom- designed DNA sequences with propensities for helix bending and folding. Interpretation of experimental results will be aided by theoretical normal mode analyses and by parallel studies of model nucleic acids for which high-resolution structures are available. The health-related impact of this program is derived from the fact that mechanisms of protein/DNA recognition will be elucidated for large biological assemblies, which are not amenable to investigation by alternative structural methods.