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Abstract
Torsional stress in linear biopolymers such as DNA and chromatin has important biological consequences. Transcription and replication of DNA twist the molecule introducing torsional stress. The accumulation of torsional stress in the molecule affects the action of enzymes that act on DNA. In higher organisms, DNA is packaged into chromatin, which alters the response of DNA to external forces. Thus, it is fundamental to have tools to measure torsional stress of DNA and chromatin.
The main objective of my dissertation is the development a tool to measure torque in single molecules at low pulling forces. This tool allows us to obtain biopolymers torsional properties. Current available techniques are not able to perform torque measurements at low pulling force and therefore are not suitable to study the torsional properties of not stretched biopolymers. I also describe a methodology to model chromatin that includes the effects of twist on its structure.
The new single molecule technique allows controlled introduction of turns into the molecule, measurement of the torque applied to the molecule with a precision of <1 pN·nm, measurement of molecule extension and the application of low pulling forces (0.1-1.5 pN). The molecule is manipulated with a cylindrical magnet and a new probe. I use the new methodology to measure torque on single DNA molecules and, for the first time, in single chromatin fibers.
The second part of this dissertation introduces a model of chromatin structure. DNA is packaged in a highly condensed structure in the nucleus of eukaryotic cells. Although chromatin structure plays an important role in regulating gene transcription, the few physical characteristics known for chromatin has made it difficult to develop realistic structural models for chromatin fibers. Using a kinematic modeling approach, I have systematically explored chromatin topology and structure. I found a large variety of highly compacted structures. Transitions between these structures to low density structures do not require introduction of turns into the chromatin fiber.
