Nynke Dekker

Abstract:

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps--ATP binding and extrusion of an initial DNA loop--allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

Abstract:

We demonstrate that the ionic current through a solid-state nanopore can be used to measure at single nanometer resolution the three-dimensional intensity profile of a laser directly in the focus of a microscope objective. We find a linear dependence of the ionic current on the incident laser power since the laser-induced heat increases the temperature locally in the solution. Our data show a temperature increase of up to 20 K in the center of the focus for a laser wavelength of 1064 nm. Measurements of the two-dimensional temperature profiles at different positions along the optical axis allow us to reconstruct the three-dimensional temperature profile of the laser focus, similar to tomography. Our new technique does not rely on the help of any optical elements and allows quantitative measurement of optical intensity or temperature distributions in aqueous environments with nanometer resolution.

Abstract:

The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.

Abstract:

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.