Abstract
This thesis describes a series of single-molecule experiments aimed at understanding the physical properties of DNA itself and the proteins that interact with it. We developed and applied sensitive techniques that allowed us to directly probe the conformation and interactions of individual DNA molecules and proteins. Magnetic and optical tweezers were used to manipulate and probe individual DNA molecules. Chapter 2 describes an improved tracking method for non-diffraction-limited objects. Chapter 3 describes the visualization of plectonemes in supercoiled DNA. Chapter 4 describes the localization of DNA bound proteins using a scanning DNA loop made using optical and magnetic tweezers. Chapter 5 describes experiments to determine the mechanical strength of the cohesin protein complex. Chapters 6–9 investigate the mechanism of DNA repair by homologous recombination. Chapter 6 addresses the conformational response of MRE11-RAD50-NBS1 complexes to nucleotide binding. Chapter 7 describes the dynamics of RecA proteins on single-stranded DNA. Chapter 8 addresses the effect of the BRCA2 CTRD domain on Rad51 filaments. Finally, chapter 9 describes experiments that probe the mechanism of homology recognition by using a dual-molecule assay.
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