Towards understanding molecular, sub-molecular and atomic mechanisms of facilitated target search on DNA
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Transcription factors and DNA repair enzymes find their target sites among billions of nonspecific sites on the genomic DNA. The mechanisms that allow proteins to rapidly scan DNA have been the subject of considerable interest in molecular biophysics since the early 1970s. However, experimental and theoretical limitations have precluded the field from advancement in some key issues. For example, intersegment transfer on DNA scanning, interplay of distinct DNA scanning mechanisms, and trapping by nonspecific DNA sites and those similar to the target sequences are not well understood. As a result, the current knowledge of DNA-scanning mechanisms remains qualitative and conceptual. The overall goal in my dissertation work was to address this problem by quantitative characterization of DNA-scanning mechanisms. Through the biophysical studies of DNA scanning by the inducible transcription factor Egr-1, we sought answers to the following fundamental questions: What dictates the efficiency in DNA scanning? Can we modulate it? What are the atomic determinants of target DNA search? This work has provided quantitative insights into the DNA-scanning mechanisms and their interplay. Our data suggest that intersegment transfer is a major pathway of DNA scanning by Egr-1, while nonspecific DNA interactions are primarily responsible for strong attenuation of target search. We found that the domain dynamics of Egr-1 plays an important role in DNA scanning, and that by modulating the dynamics, it is possible to alter the kinetic properties of the protein. We also gain some insights into how Egr-1 displaces other proteins from its target DNA. Lastly we developed NMR tools to characterize DNA scanning at atomic level. Our methodology is applicable to other DNA binding proteins. Our work has significantly improved theoretical and experimental framework for investigating mechanisms of DNA scanning by proteins, and thereby advanced our understanding of molecular and sub-molecular mechanisms of DNA scanning by proteins.