Structure and Relations of Protein and Nucleic Acids - admin supplement for the purchase of a pulsed laser system

University Of Oregon, Eugene OR

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Abstract

Summary/Abstract for requested administrative supplement to GM-15792-51. With this administrative supplement proposal to our current NIGMS grant GM-15792-51 we seek funds to purchase an additional pulsed laser system to extend and enhance our DNA replication studies. A substantial part of our ongoing research efforts are focused on achieving a detailed mechanistic understanding of the sub- assemblies of the T4 DNA replication complex by applying spectroscopic methods that site-specifically place fluorescent and optically active probes into the DNA framework of the complex. We use these labeled constructs to spectroscopically monitor ? both with bulk (ensemble) solution measurements and at the single- molecule level ? functionally significant conformational changes that occur at and near the local probe positions. The two-dimensional fluorescence spectroscopy (2DFS) method developed by Marcus can be used to determine the detailed spatial relationships between the probe chromophores in much the same way that EPR and NMR methods determine the spatial relationships between spin-label probes in molecules. We have pursued two different fluorescent probe-labeling strategies for our studies on DNA and DNA-protein interactions. We use pairs of spectrally visible Cy3 dyes placed into the DNA backbones at defined positions to study the structure and dynamics of local DNA backbone conformations, while pairs of fluorescent nucleic acid base analogues that have been substituted for the equivalent natural DNA bases are used to obtain complementary information about the structure and dynamics of the stacking and hydrogen-bonding interactions of bases and base-pairs at defined sites within our model DNA constructs. We have thus far achieved considerable success performing our 2DFS experiments using the visible Cy3 dye- substituted DNA constructs. However, the complementary experiments with fluorescent base-analogues have proceeded more slowly. This is due in large part to the absence of a dedicated pulsed laser system in our laboratory to produce the required spectrally broad (and temporally short) optical pulses centered at a wavelength of ~ 350 nm that are needed to perform 2DFS experiments on base-analogue-substituted DNA constructs. Our existing pulsed laser system is presently optimized to perform experiments on the Cy3 backbone labels, and it is not possible to modify the system on a routine basis to perform the necessary complementary experiments on base analogues. To permit us to pursue these two sets of experiments in parallel in an efficient manner we herewith request funds to purchase an additional instrument that will be optimized to perform 2DFS experiments on fluorescent base analogue substituted DNA constructs. The information obtainable in this way will greatly expand our ability to understand the DNA conformational changes that are centrally involved in regulating DNA replication systems.