Single-molecule cellular biophysics sheds new light on machines that remodel DNA

26 October 2012

In new research1 published in the journal Science, pioneering single-molecule biophysics has been used to probe the mode of action of nanoscale molecular machines that remodel DNA inside living cells, from collaborative research involving Mark Leake’s team in the Condensed Matter Physics sub-dept of Oxford Physics and David Sherratt and co-workers in Oxford Biochemistry.

Using novel, cutting-edge super-resolution optical microscopy, the researchers report on how this multi-component protein machinery can use chemical energy to dynamically adjust its stoichiometry in the presence of cellular DNA. These results shed new light on a ubiquitous family of natural molecular machines that have an essential function in the management of each cell’s genetic material, and suggests that their mode of action is very similar to that of a whole range of molecular motors that have been characterized previously using single-molecule biological physics.

Conventional biophysics methods are too slow and imprecise to monitor nanoscopic cellular machines at the single-molecule level2. To investigate individual machines one molecule at a time in single living cells, Mark Leake and co-workers have a developed a novel fluorescence imaging technology called “slimfield” in which the shape of a laser light excitation field is manipulated at the level of a single live-cell sample using home-built optical microscopy to generate very high localized intensities. This allows super-resolution molecular imaging to be performed at the millisecond time scale. State-of-the-art genetics technology allows fluorescent dye tags in the form of “fluorescent proteins” to be fused directly to the DNA which code for these remodelling machines, in effect resulting in a single molecule dye tag for each sub-unit of these molecular machines with 100% labelling efficiency. By using different forms of fluorescent protein it was possible to label different machine components simultaneously in either a green, red or yellow coloured label. In this way, “live-cell biochemistry” could be performed by monitoring the localization of these components in the slimfield microscope around the cell, and using advanced Fourier spectral analysis to convert photophysical data from the dye tags into equivalent numbers of protein molecules.

Performing these experiments on normal and mutated cell strains demonstrated that DNA remodelling machines function in a similar way to a rock-climber reaching for a handhold, in having one end anchored to a portion of cellular DNA while the other end opens and closes stochastically by converting the chemical energy stored in the universal energy currency molecule of “ATP” to mechanical “grabbing” attempts to sequester more free DNA. Experiments of this nature provide exceptional insight into vital cellular processes, and require highly constructive, long-term and sustained funding interaction between world-class Biological Physics and Biochemistry research teams, and are likely to revolutionise the future of Biomedical Science at the cellular level in our new understanding of the causes of debilitating diseases in humans.

References:

  1. Badrinarayanan A, Reyes-Lamothe R, Uphoff S, Leake MC & Sherratt DJ. In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338, 528-531 (2012)
  2. Single-molecule cellular biophysics. Leake MC. Cambridge University Press. 1st Ed. (2012) ISBN:9781107005839 www.cambridge.org/gb/knowledge/isbn/item6844254/