Tony Lemos - Thesis Dissertation Defense

In partial fulfillment of the requirements for the degree of 

Doctor of Philosophy in Physics 

School of Physics Thesis Dissertation Defense

Tony Lemos

Dr. Harold Kim, School of Physics, Georgia Institute of Technology (Advisor)

Committee members:

Dr. J.C. Gumbart, School of Physics, Georgia Institute of Technology

Dr. Zeb Rocklin, School of Physics, Georgia Institute of Technology

Dr. Peter Yunker, School of Physics, Georgia Institute of Technology

Dr. Francesca Storici, School of Biological Sciences, Georgia Institute of Technology

Abstract:

DNA is a dynamic molecule that undergoes physical manipulations by proteins. For instance, it experiences extreme bending when packaged into nucleosomes and undergoes strand separation (dehybridization) during transcription. Single-molecule assays offer a powerful approach to uncover the behavior of DNA during these processes. This dissertation presents two investigations utilizing single-molecule techniques to explore the anisotropic bending of DNA and the effects of overhangs on dehybridization kinetics.

The first investigation examines the extreme bending mechanics of DNA by directly probing the anisotropic nature of DNA minicircles through the detection of poloidal bias. We designed an assay using atomic force microscopy to map the orientation of these minicircles. By precisely monitoring the positions of protein-bound markers, we directly visualized their poloidal orientation and successfully demonstrated a sequence-dependent poloidal bias in 105 bp minicircles. Our findings were further corroborated by coarse-grained simulations.

The second investigation probes the effects of overhangs on the dehybridization kinetics of a DNA probe. Using single-molecule fluorescence resonance energy transfer, we found that the terminal base of the overhang can dictate dehybridization via base stacking. Furthermore, we identified a length-dependent relationship between the overhang and duplex stability, showing that shorter overhangs suppress dehybridization. We also found that when the overhang can form a base pair with itself, the overall stability of the bound DNA probe is significantly enhanced.