Many genetic processes require enzymes or enzyme complexes that interact simultaneously with distant sites along the genome. Such long-range DNA-enzyme interactions are important for example in gene regulation, DNA replication, repair and recombination. In addition many restriction enzymes depend on interactions between two recognition sites and form therefore a model system for studying long-range communications on DNA. Topic of the present work are Type III restriction enzymes. For these enzymes the communication mechanism between their distant target sites has not been resolved and conflicting models including 3D diffusion, 1D translocation and 1D diffusion have been proposed. Also the role of ATP hydrolysis by their superfamily 2 helicase domains which catalyse functions of many enzyme systems is still poorly understood. To cleave DNA, Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. This process strictly depends on ATP hydrolysis. The aim of this PhD thesis was to elucidate this long-range communication. For this a new single molecule assay was developed using a setup combining magnetic tweezers and objective-type total internal reflection fluorescence microscopy. In addition of being able to mechanically manipulate individual DNA molecules, this assay allows to directly visualize the binding and movement of fluorescently labelled enzymes along DNA. Applying this assay to quantum dot labelled Type III restriction enzymes, a 1D diffusion of the enzymes after binding at their target sites could be demonstrated. Furthermore, it was found that the diffusion depends on the nucleotide that is bound to the ATPase domains of these enzymes. This suggested that ATP hydrolysis acts as a switch to license diffusion from the target site which leads to cleavage. In addition to the direct visualization of the enzyme-DNA interaction, the cleavage site selection, the DNA end influence (open or blocked) and the DNA binding kinetics were measured in bulk solution assays (not part of this thesis). The experimental results were compared to Monte Carlo simulations of a diffusion-collision-model which is proposed as long-range communication in this thesis.

In the past decades, numerous single-molecule techniques have been developed to investigate individual bio-molecules and cellular machines. While a lot is known about the structure, localization, and interaction partners of such molecules, much less is known about their mechanical properties. To investigate the weak, non-covalent interactions that give rise to the mechanics of and between proteins, an instrument capable of resolving sub-nanometer displacements and piconewton forces is necessary. One of the most prominent biophysical tool with such capabilities is an optical tweezers. Optical tweezers is a non-invasive all-optical technique in which typically a dielectric microsphere is held by a tightly focused laser beam. This microsphere acts like a microscopic, three-dimensional spring and is used as a handle to study the biological molecule of interest. By interferometric detection methods, the resolution of optical tweezers can be in the picometer range on millisecond time scales. However, on a time scale of seconds—at which many biological reactions take place—instrumental noise such as thermal drift often limits the resolution to a few nanometers. Such a resolution is insufficient to resolve, for example, the ångstrom-level, stepwise translocation of DNA-binding enzymes corresponding to distances between single basepairs of their substrate. To reduce drift and noise, differential measurements, feedback-based drift stabilization techniques, and ‘levitated’ experiments have been developed. Such methods have the drawback of complicated and expensive experimental equipment often coupled to a reduced throughput of experiments due to a complex and serial assembly of the molecular components of the experiments. We developed a high-resolution optical tweezers apparatus capable of resolving distances on the ångstrom-level over a time range of milliseconds to 10s of seconds in surface-coupled assays. Surface-coupled assays allow for a higher throughput because the molecular components are assembled in a parallel fashion on many probes. The high resolution was a collective result of a number of simple, easy-to-implement, and cost-efficient noise reduction solutions. In particular, we reduced thermal drift by implementing a temperature feedback system with millikelvin precision—a convenient solution for biological experiments since it minimizes drift in addition to enabling the control and stabilization of the experiment’s temperature. Furthermore, we found that expanding the laser beam to a size smaller than the objective’s exit pupil optimized the amount of laser power utilized in generating the trapping forces. With lower powers, biological samples are less susceptible to photo-damage or, vice versa, with the same laser power, higher trapping forces can be achieved. With motorized and automated procedures, our instrument is optimized for high-resolution, high-throughput surface-coupled experiments probing the mechanics of individual biomolecules. In the future, the combination of this setup with single-molecule fluorescence, super-resolution microscopy or torque detection will open up new possibilities for investigating the nanomechanics of biomolecules.