This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
In this CAREER proposal the PI, proposes to study the target-site search strategy of DNA-binding proteins. The PI will develop a reaction-diffusion methodology to model the facilitated diffusion of a protein finding its target site. The reaction-diffusion theory will capture both the protein dynamics and the conformational behavior of the DNA, building a prediction for protein translocation that can be adapted to different physical models of DNA. The theory will be extended to 2-site binding proteins by combining facilitated diffusion to lock in the first binding site and a subsequent looping mechanism to bind the second site. The PI proposes a two-state mechanism for complex formation in 2-site binding proteins that includes thermal induced looping and a cinching mechanism due to non-specific binding of DNA to the second protein binding site. Analytical theory and Brownian dynamics simulations of supercoiled DNA will be used to develop a theoretical framework to predict the nucleation and growth of supercoils in plasmid DNA, which will be used to address the impact of plectoneme formation and restructuring in the binding kinetics of 1-site and 2-site binding proteins. In conjunction with the theoretical efforts, an integrated experimental program to test the theoretical predictions and guide model development will be developed. Using restriction enzyme digestions as a probe for target-site localization, experiments will be performed to verify the theoretical predictions and the proposed mechanisms for the target-site search. This research proposal represents a multi-faceted approach to understanding how proteins exploit non-specific binding to enhance kinetics, which is central in the biology of small numbers. Outreach efforts include the development and implementation of teaching modules into the education of high school students that are being treated for childhood cancer. The PI will also pair with a high school teacher during the summers to receive feedback on the ease of implementation and student understanding of the teaching modules.
