Untersuchung des DNA-Schneidemechanismus von Typ l Restriktionsenzymen
Zusammenfassung der Projektergebnisse
Restriction enzymes are the main defence mechanism of bacteria against invading viruses. They recognize viral DNA upon the methylation state of their target sequence and destroy it by cleaving it into pieces. Among the known restriction systems, the Type I restriction enzymes are one of the most complex systems. They consist of a methyltransferase core unit (MTase), responsible for DNA binding, which can bind up to two motor subunits (HsdRs). On viral, i.e. unmethylated DNA target sites, these motor subunits start to bind and translocate adjacent DNA in an ATP-driven manner, which leads to the formation of large DNA loops. Cleavage occurs randomly, distant from the target sites and is thought to occur upon collision with a second translocating enzyme. Aim of the project was to directly visualize the cleavage process to gain insight into the details of the collision model. For this a magnetic tweezers setup was combined with single molecule fluorescence in total-internal-reflection-fluorescence geometry (TIRF). The magnetic tweezers allowed to hold the DNA laterally stretched, while the fluorescence allowed a direct observation perpendicular to the DNA. Using motor subunits that were labelled with fluorescent quantum dots, we could directly visualize their localization and movement on DNA. In addition we could observe collisions between individual motor subunits. These results provide evidence for a looping-only translocation model. DNA cleavage could however not be directly observed due to the high enzyme concentrations (100 nM) required. The developed direct observation of DNA translocating restriction enzymes provides a useful basis for future studies of DNA cleavage by ATP-driven restriction enzymes but also for other enzymes that move along DNA.
Projektbezogene Publikationen (Auswahl)
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The Interrelationship of Helicase and Nuclease Domains during DNA Translocation by the Molecular Motor EcoR124I. J Mol Biol 384(5) 1273- 1286 (2008)
E Sisáková, M Weiserová, C Dekker, R Seidel, MD Szczelkun
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Energetics at the DNA supercoiling transition. Biophys J 98 1267-1276 (2010)
H Brutzer, N Luzzietti, D Klaue, R Seidel
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Maintaining a sense of direction during long-range communication on DNA. Biochem Soc Trans 38(2) 404-409 (2010)
MD Szczelkun, P Friedhoff, R Seidel
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Efficient preparation of internally modified single-molecule constructs using nicking enzymes. Nucleic Acids Res 39(3) e15 (2011)
N Luzzietti, H Brutzer, D Klaue, FW Schwarz, S Clausing, W Staroske, R Seidel
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Nicking enzyme-based internal labelling of DNA at multiple loci. Nat Protoc 7(4) 643-653 (2012)
N Luzzietti, S Knappe, I Richter, R Seidel
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Scanning evanescent fields using a point-like light source and a nanomechanical DNA gear. Nano Lett 12(1) 473-478 (2012)
H Brutzer, FW Schwarz, R Seidel
