Fluorescence imaging of single molecule dynamics on long single stranded DNA

Recent advances in single-molecule fluorescence imaging techniques have allowed the direct observation of protein dynamics on DNA, but the progress has been largely limited to double-stranded DNA (dsDNA) or short single-stranded DNA (ssDNA). Here, a single molecule imaging approach for observing dynamics of proteins on long ssDNA of thousands of nucleotides in length is presented, with a detailed explanation on how to implement, operate and calibrate the hybrid instrument combining single-molecule fluorescence and force spectroscopy by which we could visualize the dynamics of proteins interacting with a long ssDNA similar in length to what’s generated during DNA metabolic processes. Also, various single-stranded DNA synthesis methods are included. The ultimate goal is to study many different proteins binding to the same long ssDNA and carrying out their function in coordination with each other. As a step toward the goal, here, the applicability of our methods to the dynamics of such ssDNA-interacting proteins is demonstrated by visualizing the dynamics of the following three proteins: (1) the DNA homology search on Escherichia coli (E. coli) RecA filaments formed on ssDNA, (2) the unidirectional motion of E. coli UvrD helicase and (3) the diffusion of E. coli single-stranded DNA binding protein (SSB). We observed that the homology search of dsDNA on RecA filament is comprised of pauses at certain sites followed by fast transitions between the sticky sites via 1D diffusion. As for UvrD, with the multidimensional data obtained with our platform, we could capture the entire sequence of binding, translocation, unwinding initiation of UvrD helicase with single molecule resolution. With SSB, we found that the diffusion coefficient is three orders of magnitude higher than what was determined from SSB diffusion on short ssDNA suggesting that on long ssDNA that mimics physiological setting, SSB can migrate via a long range intersegmental transfer. Force dependence of diffusion further supports the interpretation.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2013

Author dc:creator

• Lee, Kyung Suk

Contributors dc:contributor

• Ha, Taekjip
• Chemla, Yann R.
• Dahmen, Karin A.
• Stack, John D.

• Fluorescence
• Optical Trap
• Iamging
• single stranded DNA (ssDNA)

Statement dc:rights

• Copyright 2012 Kyung Suk Lee

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/42452

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/42452