Studies of Single DNA-Histone Binding Events Using a Novel Magnetic Tweezer

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Studies of Single DNA-Histone Binding Events Using a Novel Magnetic Tweezer

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Title: Studies of Single DNA-Histone Binding Events Using a Novel Magnetic Tweezer
Author: McAndrew, Christopher Paul
Abstract: How do proteins, specifically histones, react to low levels of applied tension? Until now, low-force, single-molecule studies of DNA-protein interactions have been hampered by experimental limitations in the devices used to observe protein binding and unbinding events. To overcome this, I report on the development of a new magnetic force transducer or "tweezer" that can apply pico-Newton forces on single DNA molecules in the focus or horizontal plane and allow for the observation and measurement of single DNA-protein interaction events. This semi-closed, horizontal magnetic tweezer allows us to gather high resolution data on changes in DNA's end-to-end extension because these changes are coplanar with the pulling force. DNA constructs (lambda-DNA end labeled with a 3 <&mu>m polystyrene bead and a 2.8 <&mu>m paramagnetic sphere) and appropriate buffer (1X Tris EDTA) are introduced to a small volume semi-closed cell created using two thin cover-slips and 1mm glass spacers. This semi-closed configuration isolates a sample and produces low-noise force and extension measurements. With this new instrument, I report on the successful study of nucleosomal kinetics and energetics. Observation of individual nucleosome ruptures at and above a critical force of 1.6pN is first reported here. I claim that the capability of the instrument to produce low force-loading rates on DNA with bound proteins is responsible for this observation. This capability also allowed me to make a comparative study of nucleosomes formed using both native (typical to normal physiological conditions) and post-translationally - hyper-acetylated - histones. The experimental technique developed allowed for the observation of unbinding events at low-forces and the subsequent calculation of the differences in the binding affinities of both native histones and post-translationally modified histones.
Description: Degree awarded: Ph.D. Physics. The Catholic University of AmericaThis dissertation can be viewed by CUA users only.
URI: http://hdl.handle.net/1961/10139
Date: 2012-02-15


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