Single Molecule Studies Of Chromatin Dynamics And Transcription Coupled Repair
Biological systems create designs that respond to the need to perform specific functions. In particular, protein-DNA complexes form unique structures to maintain the stability of genetic information and yet the dynamics for necessary cellular processes. Motor proteins translocate along, and rotate around, DNA molecules to separate DNA strands, carry out polymerization reactions, resolve topological issues, repair DNA damage, and modify DNA-binding proteins. By investigating one molecular complex at a time, single molecule techniques provide controlled and quantitative approaches to measure and manipulate the protein-DNA interactions as well as visualize the function of motor proteins in real time. These techniques have now made it possible to address many problems that are difficult or impossible to study with traditional assays In this dissertation, we first introduce DNA unzipping as a powerful tool to study protein-DNA interactions at the single-molecule level. In particular, we detail protocols for preparing an unzipping template, constructing and calibrating the instrument, and acquiring, processing, and analyzing unzipping data. We also summarize major results from utilizing this technique in the studies of nucleosome structures and dynamics. After that, we use DNA unzipping to systematically investigate the interplay between nucleosome remodeling and the binding of transcription factors. The results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes. In the last chapter, we elaborate the single molecule unzipping tracker technique and its application in understanding the function of the bacterial transcription coupled repair factor Mfd. The results provide important insights into the role of Mfd beyond the scope of transcription coupled repair and significantly contribute to the understanding of Mfd function in the larger context of transcription.
Single Molecule; Chromatin; Transcription
Wang,Michelle D; Crane,Brian; Lin,Hening
Chemistry and Chemical Biology
Ph. D., Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis