Many biological functions depend on interactions between DNA and site-specific DNA- binding proteins, which must locate their specific target site within a high excess of non-specific sites. Several proteins make use of a process called facilitated diffusion, which is based on translocations of the protein along non-specific DNA and accelerates target site location. Different mechanisms involved in facilitated diffusion, such as slid-ing, hopping or jumping and intersegment transfer, have been discussed. Type II re-striction enzymes are model systems for studying protein-DNA interactions and their target search mechanism has been investigated intensively by bulk ensemble as well as by single-molecules experiments. Two open questions related to the sliding (linear diffu-sion) mechanism of Type II restriction enzymes have been addressed in this work.The first aim of this work was to investigate whether the sliding mechanism of Type II restriction enzymes is coupled to rotation. To this end, different variants of an elongat-ed restriction enzyme, consisting of EcoRV and the quasi-linear protein scRM6, have been prepared. Two important prerequisites for the visualization of rotational motion are: a preserved activity and a correct spatial orientation. Binding- and cleavage experiments demonstrated that the activity could be preserved, although being reduced by one to two orders of magnitude compared to EcoRV alone. FRET measurements however re-vealed that the extension component scRM6 is not maximally stretched out and hence the spatial orientation is not optimal. Rotational movement of the fusion protein scRM6-EcoRV could successfully be demonstrated indirectly by measuring the linear diffusion coefficient (D1) using single-molecule experiments and comparing it to theoretical mod-els. However, direct visualization of rotational movement using super-resolution micros-copy has not been possible due to suboptimal spatial orientation of the fusion protein and speed and randomness of the diffusional motion.The second aim of this work was to investigate whether the structure of a restriction enzyme influences the degree of sliding. To this end, the structurally different Type II restriction enzymes PvuII, EcoRV and BsoBI were compared regarding their confor-mation in complex with DNA and their ability to make use of linear diffusion. It was hypothesized that the more open the enzyme-DNA complex, the less linear diffusion is used by the enzyme. FRET measurements were performed to elucidate the conformation of the restriction enzyme in complex with non-specific DNA (sliding conformation), especially the extent of opening of the DNA-binding cleft. The measurements revealed that PvuII adopts and open conformation for sliding, whereas EcoRV and BsoBI adopt a more closed conformation. Conventional kinetic methods were performed to investi-gate the linear diffusion behavior and revealed that linear diffusion is used most effec-tively by BsoBI to speed up target site location and less effectively by EcoRV and PvuII. Additionally, it could be demonstrated that the ionic strength dependence of linear diffusion is different for the three enzymes. Enzymes with an open conformation (PvuII) are more affected by salt than enzymes with a more closed conformation (EcoRV, BsoBI). The results of the conformation analysis could be perfectly correlated with the results of the linear diffusion analysis and confirmed the hypothesis that re-striction enzymes with a relatively open DNA-binding cleft make less use of linear dif-fusion than enzymes with a more closed DNA-binding cleft. The hypothesis could be further confirmed by studying the linear diffusion behavior of enzyme variants in which a defined structural change had been introduced.
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