Summary/Abstract for requested administrative supplement to GM-15792-52. With this administrative supplement proposal to our current NIGMS grant GM-15792-52, we seek funds to purchase a replacement pulsed laser system to sustain 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 conformation changes that occur at and near the local probe positions. The two-dimensional fluorescence spectroscopic (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. Although we have achieved considerable success in performing our 2DFS experiments using the visible Cy3 dye-substituted DNA constructs, the laser system that is dedicated to these studies is over 15 years old, and major components of the apparatus are now beginning to fail. While the source laser is currently still operational, it is so outdated that the manufacturer no longer supports its maintenance. Failure of any of its (proprietary) electronic drivers and/or optical components would put this apparatus `out of business.' Furthermore, the various components of the instrument require major daily investments in graduate student and postdoc time to tune and stabilize the equipment ? time that could be better used to perform additional experiments. For the above reasons, we this year request funds to purchase replacement components for this instrument, which will greatly reinforce our ongoing experiments on the Cy3 dye-substituted DNA constructs, and permit these measurements to be performed in parallel with our proposed 2DFS studies of the base analogue probe- substituted DNA constructs. The information that should be obtainable by these two complementary approaches will greatly expand and speedup our ability to understand the DNA conformational changes that seem to be centrally involved in regulating DNA replication systems.
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