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.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:26229 |
| Date | 07 November 2012 |
| Creators | Schwarz, Friedrich |
| Contributors | Seidel, Ralf, Diez, Stefan, Howard, Jonathon, Schwille, Petra, Szczelkun, Mark D., Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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