Ralf Seidel

Single molecule investigations of DNA motors

Previous and current research

Translocation of enzymes along DNA, driven by molecular motors, plays a crucial role during many vital DNA-processing steps in the cellular cycle, including DNA repair, recombination, replication, transcription and restriction. These enzymes use the energy stored in ATP or other NTPs and convert it into directional motion along their template. Very often motion does not just occur just by itself but is accompanied by reshaping or processing the DNA template. For example, RNA polymerase moves along DNA and produces a transcript RNA of the transcribed gene. Helicases are splitting the DNA double helix into its single strands in a processive translocation-coupled manner. Understanding the mechanisms how DNA motors couple the ATP hydrolysis to achieve their function (motion, DNA processing) will help to gain much deeper insight in the cellular processes, which are driven by these motors.

In our biophysically oriented group we study DNA translocating motors on the level of a single molecule. Recent advances in single-molecule techniques allow to follow the action of a single molecular motor in real time. We apply magnetic tweezers, which are an excellent tool to study the translocation of single enzyme complexes on DNA. They allow to stretch and twist a single DNA molecule and to measure its end-to-end distance in real-time, while it is getting processed by an enzyme. If a DNA translocating motor pulls in DNA, its end-to-end distance changes and can thus be monitored. This way one can easily measure speeds, translocation distances, forces and torque produced by a single motor. Modeling of the measured speed as a function of generated force and ATP can reveal during which step of the ATP hydrolysis cycle force is produced. Furthermore, the behavior of the motor at special DNA sequences/structures can directly be monitored.

Current research projects involve dsDNA motors, which belong to the Super family 2 of helicases (e.g. Type I and Type III restriction enzymes) as well as viral helicases, which function in DNA replication.

Future prospects and goals

Future goals are to improve the resolution and capabilities of magnetic tweezers in order to probe and measure currently unresolved features and quantities. For example, it is aimed to resolve the individual forward steps of a DNA motor directly during its way along the DNA. Furthermore magnetic tweezers shall be coupled with other techniques like single-molecule fluorescence, which for example would allow to observe a single motor on DNA directly.

Beyond technological improvements we aim to investigate more complex biological systems like the interplay of multiple motors/proteins, e.g. at a replication fork.

Artistic view on an enzyme motor pulling on DNA in a magnetic tweezers setup

About

Selected publications

Stanley LK*, Seidel R*, van der Scheer C, Dekker NH, Szczelkun MD, Dekker C. (2006) When  a helicase is not a helicase: ds DNA tracking by the motor protein EcoR124I. EMBO J, 25: 2230-9. (* shared first author)

Seidel R, Bloom JGP, van Noort J, Dutta CF, Dekker NH, Firman K, Szczelkun MD, Dekker C. (2005) Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. EMBO J, 24: 4188-97.

Seidel R, van Noort J, van der Scheer C, Bloom JGP, Dekker NH, Dutta CF, Blundell A, Robinson T, Firman K, Dekker C. (2004) Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I. Nat Struct Mol Biol, 11: 838-43.

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