UV Resonance Raman and Fluorescence Spectroscopic Investigations of DNA Structure and Protein-DNA Complexes

Wesleyan University, Middletown CT

Investigators

Abstract

Many processes involving nucleic acids, such as replication and transcription, involve the formation of complex structures, wherein the DNA is forced to adopt a looped or bent shape because of the binding of different proteins. This project focuses on the interaction of proteins, required for forming looped DNA structures, with DNA in order to determine the contacts that are important for forming the looped structures and the energy needed to form the loop. A central question of this research is whether DNA structure plays a role in protein binding and recognition in these complex assemblies. Two different methods are used to study the interactions: fluorescence spectroscopy and a laser technique, UV resonance Raman spectroscopy. Using the fluorescence method, the nature of the complexes and the overall architecture of the complexes will be examined. This is accomplished by two different types of fluorescence experiments: anisotropy to measure local DNA flexibility and binding affinity; and resonance energy transfer to measure the relative distance between specific sites on the proteins and the DNA. The Raman technique monitors the internal vibrations of molecules, which are a sensitive measure of the structure and bonding of a molecule. Using this technique, different parts of the protein or the DNA can be selectively examined, which allows direct investigation of the important regions and contact points. These experiments probe the complexes formed between regulatory proteins and genes and contribute towards current understanding of how genomic processes are regulated in the cell. Broader impacts: Teaching and training of undergraduate and graduate students are the main educational impacts that result from this work. The project will foster an environment that promotes the integration of research and education at all levels, including the classroom. Several of the courses at the grantee institution will draw directly from research in this project. An additional impact stems from the participation of underrepresented groups in the research and from a commitment to the advancement of women in science. The research infrastructure of Wesleyan University is enhanced by this work, which involves the building of a multi-user time-correlated single photon counting instrument for fluorescence lifetime studies. This instrumentation housed at Wesleyan will be available to local scientists.