DNA tethering characterization, enzyme-mediated DNA looping under tension, and nucleosome stability in the force measuring optical tweezers
The force-measuring optical tweezers has afforded scientists unprecedented insights into many of the DNA-protein interactions that regulate genetic processes. This dissertation details the construction of an optical tweezers and the study of several such interactions. A method for generating arbitrary DNA sequences for use in the tweezers is described, including a characterization of tethering as a function of buffer pH, salinity, and DNA loading. The strength of the DNA-tethering molecular bond was measured via the distributions of unbinding times under tension. The interaction of DNA with restriction endonucleases (REases) that require two recognition sites provided a model system to study DNA looping, which is critical to such biological processes as transcription, replication, and recombination. Here it is shown that a few piconewtons of applied force completely inhibited cleavage for fifteen known or suspected two site REases, whereas it had little effect on one site REases. The application of higher tensions either inhibited or catalyzed the activity of one site REases, depending on the protein induced bend in the DNA. By replacing the Mg++ necessary for cleavage with Ca++, the loops were stabilized with the DNA intact. In the tweezers each of these loops was pulled apart, yielding a length and disruption force. Distributions thereof for the same fifteen two site REases revealed strong enzymatic effects on DNA looping. One such enzyme, Sau3AI, was used to study looping as a function of time and applied tension. Comparisons with theories indicate that protein induced bridging and kinking play a profound role in tensioned DNA looping. The extreme sensitivity of looping to tension provides a mechanism by which even extracellular stress may act as a molecular switch. Lastly, arrays of nucleosomes were assembled on arbitrary DNA using an ATP-dependent enzymatic system and then stretched in the optical tweezers. Abrupt events releasing ∼ 55 to 95 bp of DNA at forces ranging from ∼ 5 to 65 pN were observed, attributable to the unraveling of nucleosomes. The rewrapping of nucleosomes was occasionally observed upon relaxing the DNA. The unraveling and rewrapping of nucleosomes under tension may have an influence on DNA-directed cellular processes.