DNA is more than just a blueprint – it is a dynamic molecule with physical and mechanical properties that are significant for its biological functions. At any given time, various enzymes bind to and release from a multitude of sites on DNA in vivo. These enzymes not only bind to DNA, but many of them also mediate the formation of DNA loops. In the present work, loop formation and breakdown in single DNA molecules is explored for intrinsically curved DNA substrates. It is found that while loops form and break down on the order of minutes in these DNAs, the protein cannot be competed off of an intrinsically curved DNA substrate with a large excess of competitor DNA for on the order of a day. This is posited to be due to an additional interaction between LacI and the nonspecific DNA near its binding sites. Furthermore, effects are observed on DNA loops in single molecule experiments when DNA outside the looping region is altered. To explain these observations, another interaction is hypothesized in which flanking DNA associates with LacI while in the looped state. Thus, the experimental results of this work reveal two additional protein-DNA interactions in a repressor protein (i.e. LacI) which is a bacterial paradigm of gene regulation. The modified kinetic schemes which follow from these newly hypothesized interactions reveal the robust nature of protein-DNA interactions and motivate future experiments with LacI. Furthermore, it is found that altering a mechanical property (i.e. intrinsic curvature) on a DNA substrate is observed to significantly alter the kinetics of biomolecular interactions between DNA and protein.
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Changing the DNA Sequence Alters the Kinetics of Protein-Mediated DNA Looping.
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