Single molecule analysis

The concept of single-molecule analysis with Type I R-M enzymes was to make use of a Magnetic Tweezer Setup to study DNA-translocators, these enzymes are DNA-based molecular motors that, unlike other DNA-based enzymes such as polymerases, do not simply track along the DNA, but instead remain bound at their recognition site and move the DNA ends relative to this site (this makes them a simple nanoactuator – Figure 2). There potential use in nanodevices is made easier because they do not depend upon surface attachment of the motor to enable relative motion of the end of the DNA (as is required for a polymerase - Harada et al., 1999; Wang et al., 1998), but only surface attachment of DNA, which is a relatively simple process.

The Magnetic Tweezer Setup

Magnetic tweezers allow real time monitoring of protein DNA interactions without surface interference and with femtonewton sensitivity. This system can measure DNA displacements as low as 10nm. The magnetic bead required for these measurements will be attached to the end of the DNA through the biotin molecules incorporated into the DNA (see above). The other end of the DNA is surface attached, through incorporation of digoxygenin (DIG) into the DNA and surface coating of anti-DIG within the flowcell. The system is also able to produce negative, or positive, supercoils into the DNA, one turn at a time, through manipulation (spinning) of the magnetic bead. By using DNA molecules with single binding sites the stalling forces and translocation forces can be determined. We will also determine rate, processivity, pausing and step-size as a function of ATP concentration.

Figure 2

The beauty of this system is two-fold:
(i) Real-time measurements of translocation can be made with both R₁- and R₂-complexes.
(ii) The magnetic bead, bound to the DNA, can be physically rotated by the external magnets of the system. Provided the DNA substrate is NOT nicked, this results in the introduction of positive or negative supercoils in the DNA (dependent upon the direction of rotation). This allows us to study the effect of supercoiling on translocation as well as measure supercoiling that is induced by the translocation.

The Atomic Force Microscope

Using AFM we have recently been able to show images of DNA translocation and shown that two loops are formed from a R₂-complex (see Figure below and van Noort et al 2005) as expected. However, one unexpected result for the AFM studies was that the rate of translocation is far lower than that observed in solution (some 10-fold). Despite this problem, AFM represents the quickest method for visualising translocation with single-molecules.

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