Structure and Function of Holliday Junctions complexed With Proteins Probed by Fluorescence and UV Raman Spectroscopic Methods

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This project studies a central intermediate in the important cellular processes of DNA recombination and repair, which rely on the exchange of homologous DNA strands through a four-stranded structure or Holliday junction. Both DNA repair and recombination are vital functions of the cell, which are needed for cell proliferation and maintaining a stable and active genome. Errors in these processes can lead to the growth of tumors and a reduced life span. Protein- and ion-binding leads to a change in junction structure. This study specifically examines how proteins that are known to be involved in repair and recombination and are known to bind to junctions alter these structures. In particular, the ability of these proteins to either facilitate or suppress the exchanging of strands is addressed by studying the structures of the protein-DNA complexes. Two different methods are used to study the interactions: fluorescence spectroscopy and a laser technique, UV resonance Raman spectroscopy. Using the fluorescence method, the structure of the junction and the protein-binding sites are examined and compared. The Raman technique, which examines molecular vibrations, is used to probe the binding site of the ions. By elucidating the nature of these protein-junction complexes and whether they facilitate strand exchange, these studies address the overall mechanism of how DNA recombination occurs in the cell and the overall function of these proteins. This work studies these processes at a basic level and provides a better understanding of the mechanisms of DNA repair and recombination and specifically, the role of these proteins in maintaining a stable genome. Broader impacts that stem from the project are multifold. Teaching and training of undergraduate and graduate students is one of the main impacts of this work. The PI's academic institution is committed to fostering an environment that promotes the integration of research and education at all levels, including the classroom. Several of the courses taught by the PI draw directly from research in her laboratory. The PI actively works to increase the participation of underrepresented groups in science through her activities as chair of the Minority Affairs Committee of the Biophysical Society. These activities include but are not limited to participation in diversity workshops, guest lecturer in a summer mini-course in Biophysics geared towards underrepresented students and acting as a poster judge at conferences for minority students. In addition, the PI is committed to the advancement of women in science and participates in leadership and mentoring activities at the local and national level. The PI also participates in outreach activities such as 'Minds in Motion' at the K-12 level. The majority of these outreach activities are particularly geared towards groups that are traditionally underrepresented in the sciences.