DNA unwinds with magnetic tweezers
Single molecule manipulation of the twist in the DNA double helix can answer a host of tricky questions in molecular biology. Michael Gross reports
Take a length of rope in both hands and twist it. If you turn in the same direction in which the individual strings of the rope are wound around each other, there comes a point where the rope cannot be wound any tighter and it will try to curl up into shapes that are twisted on a larger scale. If you turn in the other direction, the fibres of the rope will detach from each other at least in one place, opening up a loop.
Now imagine you had extremely small tweezers that enabled you to do the same kind of twisting and twirling with the rope that is double-stranded DNA. As Vincent Croquette reported at a recent workshop meeting of the EPSRC-funded Nanonet (www.nanonet.org.uk), his group at the CNRS laboratory for statistical physics in Paris has in fact optimised the methodology known as 'magnetic tweezers' to allow them to do just that. Essentially, the researchers glue one end of a DNA double helix to a solid support, and the other end to a magnetic bead which they can move and turn around in an inhomogeneous magnetic field.
Making a bead spin to twist and untwist a molecular rope looks like just another demonstration of the amazing things one can do on the nanometre scale these days. But this has the extra benefit of being useful in a number of ways and molecular biologists are keen to apply the magnetic tweezers to their specific problems in DNA replication and transcription.
Essentially, all natural processes which involve DNA editing or readout require some extent of twisting. By monitoring both the rotation and the forces involved, researchers can gain unprecedented insights into these reactions. 'The most exciting result,' says Croquette, 'is that you see an enzyme working in real time.'
Croquette has already collaborated with the group of his CNRS colleague Gilles Charvin on DNA topoisomerases (enzymes that untwirl DNA by introducing a temporary cut in one strand through which the other strand can pass) 1 and with Giuseppe Lia's group at the University of Milan on the Gal repressor, which blocks transcription by tying DNA in a loop. The single-molecule studies showed that a slight untwirling of the double helix favours the binding of a histone-like protein known as HU, which then bends the weakened part of the helix such that two molecules of the repressor can close the loop. 2
At the same workshop, Ralf Seidel of the Technical University Delft (The Netherlands) presented another project using magnetic tweezers, which is a collaborative effort between the laboratories of Cees Dekker at Delft (famous for his work on carbon nanotubes) and Keith Firman at the University of Portsmouth, who organises the Nanonet workshops. These researchers are studying type I restriction endonucleases, kinds of DNA editing enzymes which are remarkable for the long distance between the DNA sequences they recognise and those that they actually cleave.
Between these two events, the enzyme must travel a length of up to several thousand base pairs along the double helix, following its helical twist. Conversely, this implies that an immobilised enzyme of this type can move a DNA double strand by the same length, while also rotating it. Using the magnetic bead approach, the researchers can now both record this movement and apply a counterforce to measure how strong and efficient the endonuclease is as a molecular motor. They have measured a translocation speed of over 500 base pairs per second.
But beyond the benefit of measuring physical data on a molecular scale, there is also the prospect of practical applications. Cees Dekker describes it as 'the first step toward a biologically based actuator. Such a device,' he says, 'could link the biological and silicon worlds.'
For anybody considering using the technique on their own molecules, the future is bright. 'We are presently collaborating to develop a commercial magnetic tweezer apparatus,' says Croquette. While the measuring technique usually requires the help of a physicist at the start, he promises that it soon becomes routine, and 'the biological skill is the most critical aspect'. Which means, if you can immobilise your molecule and stick a bead to the other end, you should also be able to twist it.
1. G. Charvin et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 9820.
2. G. Lia et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11373.