Structure and Relations of Protein and Nucleic Acids

University Of Oregon, Eugene OR

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Abstract

Summary/Abstract for requested administrative supplement to GM-15792-56. With this administrative supplement proposal to our current NIGMS grant GM-15792-56 we seek funds to purchase a two-photon excitation single molecule fluorescence microscope 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 position 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-photon excitation single molecule fluorescence (2PE-SMF) method developed by the Marcus lab can determine the relative stabilities of conformational species and monitor the kinetics of conformational transitions. We have pursued two different fluorescent probe-labeling strategies for our studies on DNA and protein-DNA interactions. We use pairs of spectrally visible iCy3 dyes placed (internally) within the DNA backbones at defined positions to study the structure and dynamics of local DNA backbone conformations, while pairs of fluorescent 6-MI base analogues, which have been substituted for natural guanine DNA bases, are used to obtain complementary information about the structure and dynamics of the bases and base-pairs at defined sites within our model DNA constructs. We have thus far achieved considerable success in performing bulk solution and single-molecule experiments using the visible iCy3-substituted DNA constructs. However, the complementary experiments with 6-MI have proceeded more slowly due to technical limitations that make spectroscopic studies in the ultraviolet regime challenging. Recently, we made considerable advancements in performing pulsed laser experiments on 6-MI in bulk solution using a two-photon excitation strategy that uses a dedicated laser that we purchased and assembled with an NIH Administrative supplement in 2018. Unfortunately, it is not yet possible to extend these experiments to the kinetics of local base conformational changes, as we have done for our visible iCy3 backbone probes. This is due to the absence of a dedicated single-molecule fluorescence microscope that can be integrated with our 2PE laser, which uses 680 nm pulsed light. Our current single-molecule systems are optimized for the visible iCy3 dyes that use 532 nm light. The existing instruments cannot be modified on a routine basis to perform the necessary complementary experiments on 6-MI. To pursue these two sets of experiments in parallel we herewith request funds to purchase an additional instrument that will be optimized to perform 2PE-SMF experiments on 6-MI-substituted DNA constructs. These experiments will greatly expand our ability to understand the DNA conformational changes that are central to regulating DNA replication.